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

Background: Altered sensory processing is a pervasive symptom in individuals with Autism Spectrum Disorders (ASD); people with Phelan McDermid syndrome (PMS), in particular, show reduced responses to sensory stimuli. PMS is caused by deletions of the terminal end of chromosome 22 or point mutations in Shank3. People with PMS can present with an array of symptoms including ASD, epilepsy, gastrointestinal distress, and reduced responses to sensory stimuli. People with PMS are often medicated to manage behaviors like aggression and/or self-harm and/or epilepsy, and it remains unclear how these medications might impact perception/sensory processing. Here we test this using zebrafish mutant shank3ab PMS models that likewise show reduced sensory responses in a visual motor response (VMR) assay, in which increased locomotion is triggered by light to dark transitions. Methods: We screened three medications, risperidone, lithium chloride (LiCl), and carbamazepine (CBZ), prescribed to people with PMS and one drug, 2-methyl-6-(phenylethynyl) pyridine (MPEP) tested in rodent models of PMS, for their effects on a sensory-induced behavior in two zebrafish PMS models with frameshift mutations in either the N- or C- termini. To test how pharmacological treatments affect the VMR, we exposed larvae to selected drugs for 24 hours and then quantified their locomotion during four ten-minute cycles of lights on-to-off stimuli. Results: We found that risperidone normalized the VMR in shank3 models. LiCl and CBZ had no effect on the VMR in any of the three genotypes. MPEP reduced the VMR in wildtype (WT) to levels seen in shank3 models but caused no changes in either shank3 model. Finally, shank3 mutants showed resistance to the seizure-inducing drug pentylenetetrazol (PTZ), at a dosage that results in hyperactive swimming in WT zebrafish. Conclusions: Our work shows that the effects of drugs on sensory processing are varied in ways that can be highly genotype- and drug-dependent.


Introduction
Altered sensory processing affects the majority (69-97%) of people with autism and is one of the core diagnostic symptoms in the Diagnostic and Statistical Manual V (Leekam et al., 2007;Tomchek and Dunn, 2007;Lane et al., 2011;Green et al., 2016;Tavassoli et al., 2016;Siper et al., 2017).Such symptoms includes hypo-and hyper-reactivity to stimuli, and sensory fixation (Robertson and Baron-Cohen, 2017).Consistent with this, genotype by symptom metaanalyses identified sensory hyporeactivity/increased-pain-tolerance in over 80% of individuals with Phelan-McDermid syndrome (PMS) (Mieses et al., 2016;Tavassoli et al., 2016;De Rubeis, 2018).PMS is a syndromic form of ASD, that can be caused by a chromosome 22 terminal deletion that encompasses the SHANK3 gene or a mutation in the SHANK3 gene specifically (Phelan and McDermid, 2012;De Rubeis, 2018).In addition to sensory hyporeactivity, SHANK3 mutations are correlated with a range of symptoms, that include epilepsy, sleep disturbances, and gastrointestinal distress (Soorya et al., 2013;De Rubeis, 2018;Frank, 2021;Smith-Hicks et al., 2021).This range of symptoms makes prescribing medications challenging (Costales and Kolevzon, 2015;Harony-Nicolas et al., 2015), with many individuals experiencing a prescription carousel: when one drug fails to maintain control of a symptom and/or side-effects become intolerable.Therefore, to achieve more effective symptom management, it is critical to better understand how medications impact the range of symptoms found in individuals with PMS.
Zebrafish provide characteristics that are ideal for studying how small molecules impact sensory-motor behaviors.Zebrafish sensory-motor circuits are established and become active a few days after fertilization because precocial behavioral development is essential for the survival of freely swimming larvae (Kimmel et al., 1974;Portugues and Engert, 2009;Fero et al., 2011;Kinkhabwala et al., 2011;Warp et al., 2012;Marques et al., 2018).Predator avoidance and prey capture require visual acuity, sensitive hearing, and multimodal sensory integration to activate the appropriate swimming circuits (Fero et al., 2011;Koyama et al., 2011).Importantly, sensory-motor deficits provide a proxy for circuit pathology, that can be used to identify neuropathological critical periods (Kozol, 2018;Sakai et al., 2018;Kozol et al., 2021).Finally, due to their small size and large clutch sizes (100-200 embryos), zebrafish can be screened in large numbers and also absorb most small molecules dissolved in the water that houses them.Therefore, zebrafish provide a vertebrate model that is poised to identify how small molecules influence sensorimotor behaviors in ASD models (Sakai et al., 2018).
To investigate how drugs impact SHANK3-associated hyporeactivity, zebrafish shank3a and shank3b (shank3ab) mutants were exposed to drugs and screened for sensorimotor behavior using a the well-established visual-motorresponse (VMR) assay (Burgess and Granato, 2007).During the VMR, sudden changes in illumination from light to dark evoke abrupt increases in swimming behavior as larvae search the well for a way to return to the light (Horstick et al. 2017); we capture the abrupt response by quantifying swimming in the first 30 seconds right after the transition to dark, referred to hereafter as reactivity, but the larvae sustain their search for the full 5 minutes, referred to hereafter as activity.shank3ab mutants exhibit both hyporeactivity and sustained hypoactivity in response VMR repeated lights-on to lightsoff transitions (Kozol et al., 2021).To determine the effects of small molecules on this sensorimotor deficit, we exposed larval zebrafish to the commonly prescribed medications risperidone (Nyberg et al., 1993;McDougle et al., 2005;Gencer et al., 2008;Lemmon et al., 2011), lithium chloride (LiCl) (Malhi et al., 2013;Verhoeven et al., 2013;Serret et al., 2015;Egger et al., 2017;Malhi et al., 2020), and carbamazepine (CBZ) (Mattson et al., 1992;Verhoeven et al., 2013;Jia et al., 2022).We also tested 2-methyl-6-(phenylethynyl) pyridine (MPEP), which normalized anxiety and striatal synaptic transmission in a shank3 mouse model (Wang et al., 2016).Lastly, we quantified swimming before and after exposure to pentylenetetrazole (PTZ), a drug used in animal models to better understand susceptibility to seizures, at doses that normally cause hyperactivity in wild type larvae (Baraban et al., 2005;Hoffman et al., 2016;Liu and Baraban, 2019).Results of the above experiments are summarized in the column entitled 'effect on VMR' in Table 1.
Below we describe the varied ways these drugs impacted the VMR sensorimotor behavior, from having no effect to suppressing or enhancing the VMR in a shank3-genotype-specific manner.

REVISED Amendments from Version 1
This revised version of our research article is greatly improved in response to valuable reviewer feedback and provides several important clarifications.These include an explanation of "reactivity" and "activity" endpoints (in both introduction and methods sections); expanded methods describing water quality, shank3 models that include mutations in both shank3a and shank3b ohnologs; relabeling in Figures 1 & 2; and reorganization so that text and figures are integrated with tables that include all statistical analyses coming just before Data availability.We feel that these changes make the research more accessible and we welcome further feedback.
Any further responses from the reviewers can be found at the end of the article Methods 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.The water in which the adult fish are housed are tested for pH and conductivity by probes that are always sampling, 'system water'.System water is tap water that goes through a water softener, a charcoal filter, and reverse osmosis membranes to make the water less hard/alkaline, remove contaminants and ions respectively.This purified water is stored on a 100 gallon storage tank and used for 10% daily water exchanges that are controlled by a solenoid.pH 7.0-8.1 and conductivity 350-800 µS are kept within range by two dosers, one with sodium bicarbonate (pH) and the other with instant ocean (conductivity).We also track room humidity and temperature on a daily basis.These values are important to track because the temperature of the water is regulated by air temperature.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).Readers should note that each model includes a mutation in both the a and the b ohnolog of the shank3 gene and therefor mutants are referred to as shank3ab; mutations in shank3abN are located near the N-terminus while those in shank3abC are located near the C-terminus of the predicted Shank3 protein product (Figure 1a).
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 Â 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.)/lightsoff(five min.)for a total of 40 minutes.All behavioral experiments were conducted between 11 am and 3 pm, with 2-5 independent trials.Behavior was analyzed by binning the raw ethovision movement data into 30 second and 5 minute bins.We then defined behaviors in the first 30 seconds after dark transitions as reactivity and behaviors sustained across the full five minutes of darkness as activity.Therefore, a statistical increase or decrease in swimming during the first 30 seconds was defined as hyperreactive or hyporeactive respectively; a statistical increase or decrease in swimming during the full five minutes was defined as hyperactive or hypoactive respectively.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 (200 mg/L dissolved in system water).

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.Both heterozygote and homozygote larvae were tested for seizure susceptibility, however to remain consistent with the other genotypes analyzed in the study, we chose to focus on the homozygote data.

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.

Results
Zebrafish shank3ab mutants are hypoactive and hyporeactive in response to lights-off transitions We previously showed that both shank3abN and shank3abC mutants exhibit sensory hyporeactivity (activity during first 30 seconds in dark) and hypoactivity (activity over full 5 minutes in dark) in a light to dark transition paradigm, the VMR assay (Kozol et al., 2021).Here we repeat this assay, but this time in the presence of the drug carrier 0.1% DMSO.In comparison to WT (Figure 1a & b, Tables 2 & 3), both shank3abN-/and shank3abC-/models exhibited hyporeactivity and hypoactivity (Figure 1c-e, Tables 4-7).These results provide a reliable sensorimotor phenotype that can be quantified following exposure to selected drugs (Kozol & Dallman, 2023).Dose-response curves to identify effective doses for each small molecule Dose-response curves for small molecules were performed to investigate how these drugs impact the VMR in WT larvae.Risperidone did not affect the VMR at 1 μM, while at 10 and 20 μM doses, the VMR was decreased (Figure 2a, Tables 8-11).LiCl did not impact the VMR in WT larvae, despite exceeding previously published concentrations (Figure 2b, Tables 12-13).In contrast, CBZ had varying effects on both reactivity and activity: 80 μM and 120 μM CBZ concentrations showed no effect a, while 200 μM caused larvae to be hypo-reactive (Figure 2c, Tables 14-17).Similarly, 1 μM of MPEP did not affect the VMR, while 5 and 10 μM the VMR was decreased (Figure 2d, Tables 18-21).These results provide the lowest effective concentrations for each drug, risperidone (10 μM), CBZ (200 μM) and MPEP (5 μM), that caused a significant decrease in WT activity and reactivity; for LiCl we proceeded with the high dose of 5 mM.We next used these small molecule concentrations to compare how each would impact sensorimotor behavior in shank3ab-/mutants.

Risperidone normalizes lights-off hypoactivity in shank3ab mutants
Risperidone is commonly prescribed in ASD for aggressive, self-injurious and hyperactive behavior (Lemmon et al., 2011).In shank3ab-/mutants, 10 μM risperidone exacerbated hyporeactivity, but normalized hypoactivity, with shank3ab mutants achieving wild-type levels of swimming over the full duration of lights-off conditions (Figure 3, Tables 22-25).These results show that risperidone both reduced shank3 stimulus reactivity, and normalized overall stimulus-driven behaviors in shank3ab mutants.
LiCl does not impact light evoked sensorimotor behavior in shank3 mutants or wildtype Lithium chloride (LiCl) has been prescribed for several neuropsychological disorders, including bipolar disorder, depression, and ASD (Malhi et al., 2020).LiCl has been prescribed to individuals with PMS that exhibit bipolar depression, psychosis, and catatonic behavior (Verhoeven et al., 2013;Egger et al., 2017).Exposure to 5 mM LiCl caused no change in shank3ab-/-VMR (Figure 4, Tables 26-29).Therefore, LiCl does not impact visual processing in either WT zebrafish or shank3 mutant larvae.
Carbamazepine does not impact light evoked sensorimotor behavior in shank3 mutants or wildtype Carbamazepine (CBZ) is commonly prescribed to control seizures in individuals with epilepsy (Mattson et al., 1992).For individuals with PMS, CBZ has been prescribed following symptom resistance to common mood stabilizers, such as lithium and valproic acid (Verhoeven et al., 2013).WT and shank3abN-/mutants VMR reactivity trended reduced with CBZ exposure but did not reach p < 0.05 (Figure 5, Tables 30-33).By contrast, shank3ab C-terminal VMR reactivity was unaffected by CBZ exposure.These results suggest that CBZ could have differential impacts on sensorimotor circuits depending on the location of the mutation in the shank3 gene.Wildtype zebrafish recapitulated shank3 mutant hypoactivity and hyporeactivity when exposed to the mGlur5 antagonist MPEP While the molecules described above have been prescribed for ASD and epilepsy, we were also interested in investigating compounds used to rescue behavioral deficits in Shank3 mouse models (Wang et al., 2016).We found that MPEP did not affect the VMR in shank3ab mutants however, MPEP was sufficient to cause hyporeactivity and hypoactivity in WT larvae (Figure 6, Tables 34-37).Therefore effects of MPEP on sensory-induced behaviors were genotype-dependent.
shank3abN and C homozygous mutants do not exhibit hyperactive swimming in response to the GABA A receptor antagonist pentylenetetrazole A standard approach used in animal models to test for susceptibility to seizures related to reduced GABAergic inhibition is to test responses to the GABA A receptor antagonist pentylenetetrazole (PTZ) (Baraban et al., 2005;Hoffman et al., 2016;Liu and Baraban, 2019).In response to 3 mM PTZ, both N and C shank3ab-/larvae fail to exhibit WT level of hyperactivity suggesting altered GABAergic signaling in the shank3ab mutant models (Figure 7, Tables 38-41).and Kim, 2000;Grabrucker, 2014;Harony-Nicolas et al., 2015;Kozol et al., 2015Kozol et al., , 2021;;Harris et al., 2016;Engineer et al., 2018;James et al., 2019;Breen et al., 2020;Lutz et al., 2020).Here we tested how drugs targeting aggressive behavior, catatonia, and/or epilepsy affect sensorimotor VMR behaviors in zebrafish shank3 models of PMS.We found that drugs were neutral, enhanced or suppressed sensory-induced behavior in a genotype-and drug-dependent manner.
Zebrafish, in particular, provide a cost-effective and high-throughput way to test how medications impact behaviors (Rihel et al., 2010;Kokel and Peterson, 2011;Rihel and Schier, 2013;Jordi et al., 2015;Bruni et al., 2016;Hoffman et al., 2016).We previously validated shank3ab N and C zebrafish models and showed a shank3ab mutant dose-dependent reduction in the VMR (James et al., 2019;Kozol et al., 2021).Because the VMR phenotype is strongest in shank3ab homozygous larvae, we focused on this genotype for our small drug screen.Widely-prescribed, mood-stabilizing medications risperidone and LiCl had distinct effects on the VMR.Risperidone exacerbated shank3 VMR hyporeactivity and rescued overall activity to WT levels; by contrast, LiCl had no effect on the VMR in any of the three genotypes tested.In addition to the beneficial effects of risperidone however, this medication is associated with weight-gain in humans and reduced gastrointestinal motility in zebrafish (de Alvarenga et al., 2017;Guber et al., 2022).Consistent with this, risperidone D 2 and 5-HT 2 receptor targets (Nyberg et al., 1993) are expressed and regulate function in both brain and gut (Taniyama et al., 2000;Eliassi et al., 2008;Feng et al., 2020).Therefore, risperidone creates known symptom trade-offs in addition to improving mood in people and visual processing in zebrafish.
Treatment-resistant epilepsy in Phelan-McDermid Syndrome is one of the most difficult symptoms to manage and also one for which there are many drug options (Chakraborty et al., 2022).CBZ, a sodium channel blocker, has been used in patients with PMS who were resistant to mood stabilizers (Mattson et al., 1992;Verhoeven et al., 2013Verhoeven et al., , 2020)).CBZ reduced reactivity to dark transitions in WT and shank3abN-/larvae (though VMRs in neither genotype reached p<0.05) but had no effect on median VMR values in shank3abC-/larvae, indicating possible shank3 allele-specific differences in the way CBZ impacts the VMR.Consistent with shank3 allele-specific differences, whole brain activity mapping in these same models showed a greater activity in mid and hindbrain circuits in response to dark transition in shank3abN than shank3abC alleles (Kozol et al., 2021).Another drug that addresses seizure susceptibility is PTZ, a GABA A receptor antagonist that is used to test seizure susceptibility in zebrafish and murine models.Our findings that shank3ab models are resistant to doses that make WT larvae hyperactive suggest that these models might have fewer GABA A receptors targets for PTZ to act upon.As with the mood stabilizers, the effects of CBZ and PTZ were both drugand genotype-dependent.
Finally, our findings that MPEP made WT behave like shank3ab-/larvae in the VMR assay suggest that blocking mGluR5 may affect sensory processing.MPEP blocks mGluR5 and improves excessive grooming and striatal synaptic plasticity in a mouse shank3 model (Wang et al., 2016).GluR5 continues to show promise as a regulator of excitatory/ inhibitory balance in the striatum where a negative correlation between mGluR5 and GABA was measured in autistic people using fMRI; mouse Cntnap2 mutants showed a similar negative mGluR5 and GABA correlation that was not found in either Shank3 or 16p11.2deletion models (Carey et al., 2022).

Summary/conclusions
Our findings highlight the genotype-, drug-, and phenotype-specific challenges of designing treatment strategies for Phelan-McDermid Syndrome.These include trade-offs that can occur when a drug like risperidone improves sensoryprocessing and mood at the expense of gut function and differential effects of drugs on different symptoms.

Sara Moir Sarasua
Clemson University, Clemson, South Carolina, USA Thank you for the opportunity to review this interesting and timely research.The authors present a study in which they use the zebrafish model of two shank3ab mutants (affecting either the N or C terminus of shank3a and shank3b) to measure sensory-motor behaviors by subjecting larvae to one of 3 medications for 24 hours and then measuring response to light/dark cycles.The authors find that risperidone, but not LiCl or Carbamazepine, affects the response to light.They also find that the shank3ab models are resistant to the seizure-inducing drug PTZ.The methods presented will be helpful to others using the zebrafish model for Phelan-McDermid syndrome related research.This research is important because it models commonly used therapies in the zebrafish model with the hope of better understanding effective treatments in people with Phelan-McDermid syndrome due to SHANK3 haploinsufficiency or SHANK3 pathogenic variants.As such it will be of interest both to researchers investigating animal models as well as clinical researchers.
With an interdisciplinary audience in mind, there are places where additional information would be most helpful.
Our specific comments follow:

Abstract:
The authors state in the Results that "risperidone normalized the VMR in shank3 models".In the Results section, the authors state that "risperidone exacerbated hyporeactivity, but normalized hypoactivity."Would the authors clarify?
include both shank3a and shank3b deletions of either the C or N terminus regions.Please direct the reader to the shank3a and shank3b model in Figure 1a.While this information is in the Figure, it is not mentioned in text.And because the figures and tables are located some distance from the text, the information is not as accessible as it could be.Please define in the methods how activity/hypoactivity was defined or measured.Please define how reactivity/hyporeactivity was defined.Does VMR include both of these measures?Or only the reactivity?

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In the "Drug Screening" section, the system water needs more detail of quality measures i.e.. breakdown of the monitored levels of alkalinity, hardness, carbon dioxide, dissolved oxygen, pH, and anything else that is measured per-period or weekly.
In the statistical analysis section, please tell the reader how the analyses were to be interpreted.By this we mean that normalization or rescue of a response was determined by comparing WT to mutant and finding no statistically significant difference, or a reduced difference.A challenge we discovered when interpreting the figures which display the statistical significance levels between WT-DMSO and mutants or WT drug and mutants, is that it was the lack of significance (lack of stars) that led to the interpretation of results.For instance, in Figure 3 describing response to risperidone, the caption says that risperidone normalizes the response.The reader can observe that the significant differences observed in the 30 second test, were no longer apparent in the 5 minute test.The 5 minute tests were similar between WT and mutants.We are so trained to look for the stars on graphs that it is hard to observe the lack of stars.Do the authors have suggestions to guide the reader in how to interpret these results?When the "biologically significant" finding is the non-statistically significant finding.It may also be of help to the reader to include a particular focus on the tables with statistical tests that much interest is given to such as the comparison of WT to mutant (showing difference) then WT to mutant-risp (showing no difference).Thus risperidone normalizing the outcome.Our first inclination was to suggest that the many tables be placed in a supplemental file, however, they are quite important.Is there a way to highlight these key comparisons that were used to come to the conclusions that the authors make?
Table 2. Please tell the reader who is in Group A and who in Group B.  Figures 3, 4, 5.The figures show statistical comparisons between WT DMSO vs. the 4 shank3ab genotypes and WT drug vs. the 4 shank3ab genotypes by indicating with stars whether the comparisons are statistically significant.However, it is the absence of a statistical difference that leads to the interpretation.This is a challenge in reading the graphs and requires a detailed review of the statistical tables.Do the authors have any suggestions?
Figure 4.In this figure caption, it reads "reactivity or activity" rather than "hyporeactivity or hypoactivity" as in the previous figure.It helps the reader to be consistent in use of the terms across the written portion as well as figures/tables.
Figure 7. panel b and c, label WT and N-/+ and N -/-.In panel a, it appears the investigators assessed N-/-and N-/+.However, in the accompanying graphics, it appears only the N-/-is graphed.Is that correct?Is N-/+ a heterozygote?I do not see assessment of heterozygotes in the methods or anywhere else in the manuscript.Please include the methods in the methods section.
Page 26.It is understandable to model the strongest phenotypic response in the homozygous models.However, PMS is caused by a heterozygous deletion/variant in SHANK3.Would the authors comment on how their results might be applied to the case of heterozygous mutations and how this work translates to the human clinical condition?Would the authors also comment on how the fish paralogs shank3a and shank3b relate to the human SHANK3?
It would help the reader if the authors would help interpret the direction of effect that is to the benefit.For instance, if treated with risperidone, the activity in the mutants is less than when not treated with risperidone.The authors say this exacerbated the hyporeactivity.For clinical usage, is increased hyporeactivity (is that less reactivity?) a desired outcome?If treatment with risperidone normalized activity, is that a desired outcome?Back to an earlier comment, it would help the reader to provide a summary table telling the reader, for each drug and each genotype, what the effect was (no difference, Decreased activity, Increased activity, Normalized compared to WT, decreased reactivity, Increased reactivity, Normalized reactivity, And which direction is the desired direction) What does this study add to what was previously known?

General points
The flow of the manuscript is disrupted with the many figures and tables and the text is sometimes lost among the tables.It would help to have the Figures and Tables placed where referenced and include them all within the Results section rather than interspersed.

Discussion
How do the authors interpret the genotype-specific findings?Response: We agree that as written there seems to be a discrepancy between what is stated in the abstract and results/discussion.We decided not to include the terms and definitions of hypoactivivity and hyporeactivity in the abstract due to the need for brevity but in the current version, we edited the text in the abstract results section to read "We found that risperidone partially normalized the VMR in shank3 models."

Introduction:
The authors provide a sound justification for the use of the zebrafish model in measuring sensory motor behavior.
The authors do not indicate why PTZ trials were included in the experiments.This should be added to the introduction.Because epilepsy and seizures are important phenotypes in PMS, it is reasonable to look at this phenotype, thus it should be included in the introduction.
Response: We edited the following sentence to more clearly introduce PTZ experiments " Lastly, we quantified swimming before and after exposure to pentylenetetrazole (PTZ), a drug used in animal models to better understand susceptibility to seizures, at doses that normally cause hyperactivity in wild type larvae (Baraban et al., 2005;Hoffman et al., 2016;Liu and Baraban, 2019).

Methods:
Page 3 (of the pdf version of the article).It would help the reader to state that these mutants include both shank3a and shank3b deletions of either the C or N terminus regions.Please direct the reader to the shank3a and shank3b model in Figure 1a.While this information is in the Figure, it is not mentioned in text.And because the figures and tables are located some distance from the text, the information is not as accessible as it could be.
Response: We agree that more information on the design of our mutations provides clarity for the reader.We have since added the text, "Readers should note that each model includes a mutation in both the a and the b ohnolog of the shank3 gene and therefor mutants are referred to as shank3ab; mutations in shank3abN are located near the N-terminus while those in shank3abC are located near the C-terminus of the predicted Shank3 protein product (Figure 1a)."Response: We thank the reviewer for finding this omission in the methods.We have added the definition of activity and reactivity, along with the measurement units that were compared.The text now reads, "Behavior was analyzed by binning the raw ethovision movement data into 30 second and 5 minute bins.We then defined behaviors in the first 30 seconds after dark transitions as reactivity and behaviors sustained across the full five minutes of darkness as activity." Please define in the methods how activity/hypoactivity was defined or measured.
Please define how reactivity/hyporeactivity was defined.Does VMR include both of these measures?Or only the reactivity?

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Response: We thank the reviewer for finding this omission in the methods.We have added the definition of hyper-and hypo-following the above mentioned definition of baseline activity and reactivity.The manuscript now reads, "Therefore, a statistical increase or decrease in swimming during the first 30 seconds was defined as hyperreactive or hyporeactive respectively; a statistical increase or decrease in swimming during the full five minutes was defined as hyperactive or hypoactive respectively." In the "Drug Screening" section, the system water needs more detail of quality measures i.e.. breakdown of the monitored levels of alkalinity, hardness, carbon dioxide, dissolved oxygen, pH, and anything else that is measured per-period or weekly.
Thank you for pointing out this oversight.We have added the following to the methods section." The water in which the adult fish are housed are tested for pH and conductivity by probes that are always sampling, 'system water'.System water is tap water that goes through a water softener, a charcoal filter, and reverse osmosis membranes to make the water less hard/alkaline, remove contaminants and ions respectively.This purified water is stored on a 100 gallon storage tank and used for 10% daily water exchanges that are controlled by a solenoid.pH 7.0-8.1 and conductivity 350-800 µS are kept within range by two dosers, one with sodium bicarbonate (pH) and the other with instant ocean (conductivity).We also track room humidity and temperature on a daily basis.These values are important to track because the temperature of the water is regulated by air temperature." In the statistical analysis section, please tell the reader how the analyses were to be interpreted.By this we mean that normalization or rescue of a response was determined by comparing WT to mutant and finding no statistically significant difference, or a reduced difference.A challenge we discovered when interpreting the figures which display the statistical significance levels between WT-DMSO and mutants or WT drug and mutants, is that it was the lack of significance (lack of stars) that led to the interpretation of results.For instance, in Figure 3 describing response to risperidone, the caption says that risperidone normalizes the response.The reader can observe that the significant differences observed in the 30 second test, were no longer apparent in the 5 minute test.The 5 minute tests were similar between WT and mutants.We are so trained to look for the stars on graphs that it is hard to observe the lack of stars.Do the authors have suggestions to guide the reader in how to interpret these results?When the "biologically significant" finding is the nonstatistically significant finding.It may also be of help to the reader to include a particular focus on the tables with statistical tests that much interest is given to such as the comparison of WT to mutant (showing difference) then WT to mutant-risp (showing no difference).Thus risperidone normalizing the outcome.Our first inclination was to suggest that the many tables be placed in a supplemental file, however, they are quite important.Is there a way to highlight these key comparisons that were used to come to the conclusions that the authors make?
Response: We agree that it might be confusing to have the lack of significance indicate rescue.We have edited the figure legend to now state, "No statistical significance between drug exposed mutant and control was interpreted as normalization." Table 2. Please tell the reader who is in Group A and who in Group B.
Response: We agree with the reviewer that these labels were arbitrary and did not define the categories analyzed.We have updated these labels to "lights-on" for "Group A" and "lights-off" for "Group B".
Table 5, 2 nd to last row.Why does it say ABTL instead of WT?Elsewhere sometimes ABTL is used and sometimes WT.Are they interchangeable?I suggest using just WT if they are the same.The methods section tells the reader that WT is ABTL.
Response: We agree with the reviewer that multiple names is confusing.We have since taken ABTL out of all instances, besides the initial definition of WT in the "Ethics, fish maintenance and husbandry" section.
Figures.We appreciate the authors defining what is depicted in the box plots and inclusion of the sample sizes.
Response: We thank the reviewer for the compliment.for distance (mm/30 sec) and the other Distance (mm/5min).It would help the reader to label the two columns/graphs.It is defined in Figure 3 and would help the reader to define it in Figure 2. It would help the reader to label each column (Activity) and (Reactivity) so the reader more clearly understands which graph shows which outcome.
Response: Thank you for this suggesting this change.We have revised both Figure 1  In terms of the relationship between human and zebrafish gene paralogs, we previously published a study that has an analysis that describes how conserved shank3 genes are in fish.We found that both ohnologs are highly conserved with humans, with both copies maintaining all of the major conserved protein interacting domains.Finally, we found shank3a was more highly conserved, with higher percent pairwise comparisons and inclusion of smaller single protein interacting domains.See Kozol et al. 2015, Human Molecular Genetics for more details.
It would help the reader if the authors would help interpret the direction of effect that is to the benefit.For instance, if treated with risperidone, the activity in the mutants is less than when not treated with risperidone.The authors say this exacerbated the hyporeactivity.For clinical usage, is increased hyporeactivity (is that less reactivity?) a desired outcome?If treatment with risperidone normalized activity, is that a desired outcome?Back to an earlier comment, it would help the reader to provide a summary table telling the reader, for each drug and each genotype, what the effect was (no difference, Decreased activity, Increased activity, Normalized compared to WT, decreased reactivity, Increased reactivity, Normalized reactivity, And which direction is the desired direction) Response: We understand that our original interpretation of the relationship between our results and how they relate to the human condition was not clear.This study was looking to see how drugs prescribed for one symptom, such as aggressive behavior or epilepsy, impacted sensory perception, with a focus on sensory hyporeactivity that is found in our models.Therefore, our results provide evidence that many non-selective molecules used to treat core symptoms, can in reality exacerbate or help medicate sensory symptoms that may not have been tested yet in a clinical study.We strongly believe that normalizing or medicating a sensory deficit to baseline should be a primary goal for a clinician, seeing that sensory deficits are one of several quality of life ailments for individuals with PMS.Finally, table 1 provides the primary results for each drugs effect on VMR and we have provided clarity in the text to direct the reader to this information.See previous response.
to CBZ?
Concentration of each drug in the bath solution was determined by making a dose response curve first in Wild type fish (Fig. 2).The concentration in the CNS may reach that of the bath solution depending on the chemical property of the drug, while some drugs may display disparity between CNS and bath.It will be difficult to directly measure the concentration of each drug in the brain.Therefore, I suggest adding some discussion on pharmacokinetics (absorption, excretion, blood brain barrier etc.) in the text.

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In Fig. 1c and d, please put labels on the horizontal axes.While readers can identify three groups based on their colors, labeling will make the identification easier.

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If allowed in the journal format, please consider moving most of the tables to supplement.
It is definitely good that readers can directly look at all the analysis.On the other hand, having 41 tables in the middle of the main text is somewhat distracting.I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
respond (our responses are in italics) to each suggestion below.Please provide the definition of "activity" and "reactivity" in Introduction or Methods.I presumed that the former corresponds to the overall activity during the dark 5 min while the latter to that of first 30 seconds.Because this concept is central to the study design and the analysis, it may be worthwhile to highlight it in Fig. 1.

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Response: We agree with the reviewer that we omitted the definitions for activity and reactivity.
We have since edited the text of both the introduction and the methods to read, Introduction: "During the VMR, sudden changes in illumination from light to dark evoke abrupt increases in swimming behavior as larvae search the well for a way to return to the light (Horstick et al. 2017); we capture the abrupt response by quantifying swimming in the first 30 seconds right after the transition to dark, referred to hereafter as reactivity, but the larvae sustain their search for the full 5 minutes, referred to hereafter as activity".Methods: "Behavior was analyzed by binning the raw ethovision movement data into 30 second and 5 minute bins.We then defined behaviors in the first 30 seconds after dark transitions as reactivity and behaviors sustained across the full five minutes of darkness as activity.Therefore, a statistical increase or decrease in swimming during the first 30 seconds was defined as hyperreactive or hyporeactive respectively; a statistical increase or decrease in swimming during the full five minutes was defined as hyperactive or hypoactive respectively" The difference of reactivity and activity between N-term and C-term mutants in CBZ is intriguing.Is it known whether human patients with either mutation show distinct response to CBZ?
○ Response: We appreciate the reviewer's curiosity in how our results relate to the literature.We know that CBZ is used to treat intractable epilepsy in some individuals with PMS.However, information regarding medication is usually a portion of meta analyses for clinical reviews or perspectives.Therefore, they do not indicate how CBZ impacts other symptoms of the patients.
Concentration of each drug in the bath solution was determined by making a dose response curve first in Wild type fish (Fig. 2).The concentration in the CNS may reach that of the bath solution depending on the chemical property of the drug, while some drugs may display disparity between CNS and bath.It will be difficult to directly measure the concentration of each drug in the brain.Therefore, I suggest adding some discussion on pharmacokinetics (absorption, excretion, blood brain barrier etc.) in the text.

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Response: Thank you for pointing this out.We have added a paragraph to the discussion: "It should be noted that the concentration of the drug reported here corresponds to that in the bath solution surrounding the larvae.This bath concentration may differ substantially from the brain concentration due to differences in on a drug by drug basis related to pharmacokinetic differences.These include the drug's ability to cross the skin and/or blood brain barrier as well as differences in kinetics of drug breakdown and excretion (Skiba et al., 2023;Windell et al., 2023).Also, this route of administration differs fundamentally from that in people and therefore drug dosage in zebrafish does not correspond to that in people." In Fig. 1c and d, please put labels on the horizontal axes.While readers can identify three groups based on their colors, labeling will make the identification easier.

Figure 1 .
Figure 1.Stable shank3ab mutant lines exhibit hyporeactivity and hypoactivity following a light to dark transition.a) shank3ab N-terminal and C-terminal mutants were designed to target regions with known deleterious mutations in individuals with PMS.b) Trace line graphs showing four cycles of 5 minutes lights-on to lights-off.Checkered boxes on the x-axis represent lights on and off.c) Lights on to off paired comparison, highlighting no significant change in activity of shank3ab N terminal mutants during the first 30 sec lights-off.d) Box plots showing first 30 sec lights-off activity.e) Box plots showing activity across the full 5 minutes lights-off.Box plots represent 25 th and 75 th percentile, and median, with min to max whiskers.Sample sizes: WT = 50, shank3 N = 65, shank3 C = 44.p values; * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001.

Figure 2 .
Figure2.Please label the 2 columns of the graph.Two graphs are shown for each drug.One for distance (mm/30 sec) and the other Distance (mm/5min).It would help the reader to label the two columns/graphs.It is defined in Figure3and would help the reader to define it in Figure2.It would help the reader to label each column (Activity) and (Reactivity) so the reader more clearly understands which graph shows which outcome.

Figure 2 .
Figure 2. Please label the 2 columns of the graph.Two graphs are shown for each drug.One

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Is the work clearly and accurately presented and does it cite the current literature?YesIs the study design appropriate and is the work technically sound?YesAre sufficient details of methods and analysis provided to allow replication by others?YesIf applicable, is the statistical analysis and its interpretation appropriate?YesAre all the source data underlying the results available to ensure full reproducibility?YesAre the conclusions drawn adequately supported by the results?YesCompeting Interests: No competing interests were disclosed.

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Response: We have added labels to the bottom of the box plots in Figure1.

Table 1 .
Drugs used in this study are listed to the left followed by indication and target(s)/mechanism of action.These are based on relevant references in the rightmost column.Drug effects on VMR in are based results from this study.

Table 8 .
ANOVA of 30-second light-off for risperidone dose response curve in WT larvae.See Figure2a.

Table 9 .
Dunn's multiple comparisons of 30-second lights-off for risperidone dose response curve.See Figure2a.

Table 10 .
ANOVA of 5-minute lights-off for risperidone dose response curve.See Figure2a.

Table 11 .
Dunn's multiple comparisons of 5-minute lights-off for risperidone dose response curve.See Figure2a.

Table 12 .
ANOVA of 30-second light-off for lithium chloride dose response curve.See Figure2b.

Table 13 .
ANOVA of 5-minute lights-off for lithium chloride dose response curve.See Figure2b.

Table 14 .
ANOVA of 30-second light-off for carbamazepine dose response curve.See Figure2c.

Table 15 .
Dunn's multiple comparisons of 30-second lights-off for carbamazepine dose response curve.See Figure2c.

Table 16 .
ANOVA of 5-minute lights-off for carbamazepine dose response curve.See Figure2c.

Table 17 .
Dunn's multiple comparisons of 5-minute lights-off for carbamazepine dose response curve.See Figure2c.

Table 18 .
ANOVA of 30-second lights-off for MPEP dose response curve.See Figure2d.

Table 19 .
Dunn's multiple comparisons of 30-second lights-off for MPEP dose response curve.See Figure2d.

Table 20 .
ANOVA of 5-minute lights-off for MPEP dose response curve.See Figure2d.

Table 21 .
Dunn's multiple comparisons of 5-minute lights-off for MPEP dose response curve.See Figure2d.

Table 1 .
It is somewhat unclear whether the "Effect on VMR" in this table derives from the cited literature or from the results of the research.After reading the manuscript, it would be helpful to have a table to summarize the results of the research in a table similar to Table1, but based just on the present research results.A lot of work and many many statistical analyses were performed in this research study, and the results get somewhat lost.A summary table with an interpretation of what they mean would be very helpful to the reader.High-throughput behavioral screens -what is the outcome measured in the VMR experiments?Is it the same as described for the PTZ trials -total distance moved for 10 minutes? ○

Table 5 ,
2 nd to last row.Why does it say ABTL instead of WT?Elsewhere sometimes ABTL is used and sometimes WT.Are they interchangeable?I suggest using just WT if they are the same.The methods section tells the reader that WT is ABTL.Figures.We appreciate the authors defining what is depicted in the box plots and inclusion of the sample sizes.

the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Yes If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes Competing Interests:
No competing interests were disclosed.

Table 1 .
It is somewhat unclear whether the "Effect on VMR" in this table derives from the cited literature or from the results of the research.After reading the manuscript, it would be helpful to have a table to summarize the results of the research in a table similar to Table1, but based just on the present research results.A lot of work and many many statistical analyses were performed in this research study, and the results get somewhat lost.A summary table with an interpretation of what they mean would be very helpful to the reader.Response: We agree with the reviewer that clarity on the content of table 1 would help direct the reader's attention to the important summaries from our study.This table includes the drug, drug classification, receptors that the drug targets, the summary of how the drug effected VMR in our study and references that provided mechanism of action and/or helped us determine dosages to test for our dose response assays.We have since edited the last sentence of the introduction to read "Results of the above experiments are summarized in the column entitled 'effect on VMR' in Table1."And the table description to read, "Drugs used in this study are listed to the left followed by indication and target(s)/mechanism of action.These are based on relevant references in the rightmost column.Drug effects on VMR in are based results from this study."Page4.What is the dosage used for the MS222 to euthanize the larvae? ○ Kozol et al. 2015statistical comparisons between WT DMSO vs. the 4 shank3ab genotypes and WT drug vs. the 4 shank3ab genotypes by indicating with stars whether the comparisons are statistically significant.However, it is the absence of a statistical difference that leads to the interpretation.This is a challenge in reading the graphs and requires a detailed review of the statistical tables.Do the authors have any suggestions?Response: We agree with the reviewer that the submitted version did not explicitly state how to interpret the graphs.We have since edited the figure legends of Figures4-5to read, "No statistical significance between drug exposed mutant and control was interpreted as normalization."Figure4.In this figure caption, it reads "reactivity or activity" rather than "hyporeactivity or hypoactivity" as in the previous figure.It helps the reader to be consistent in use of the terms across the written portion as well as figures/tables.Response: We agree that the figure is confusing when seeing the inconsistencies in genotype titles for the image and graph.We decided to focus on the homozygote data, because heterozygotes were not tested or analyzed for the other drugs used in the study.We have edited the text for the "Drug Screening" section to now include clarity about our analysis.The text now reads, "Both heterozygote and homozygote larvae were tested for seizure susceptibility, however to remain consistent with the other genotypes analyzed in the study, we chose to focus on the homozygotic data for statistical analyses to be consistent with the rest of the paper."Page26.It is understandable to model the strongest phenotypic response in the homozygous models.However, PMS is caused by a heterozygous deletion/variant in SHANK3 .Would the authors comment on how their results might be applied to the case of heterozygous mutations and how this work translates to the human clinical condition?Would the authors also comment on how the fish paralogs shank3a and shank3b relate to the human SHANK3?Response: We assessed heterozygotes in previous research for VMR and other phenotypes, seeKozol et al. 2015 Human Molecular Genetics, James et al. 2019 Molecular Autism, and Kozol et al.  2021Nature Communications Biology.In our previous work, we found that the homozygotes provided the most pronounced version of a phenotype and therefore an ideal choice for determining how drugs impact these phenotypes.Because homozygotes are viable in zebrafish, we believe the model provides an opportunity to screen a relatively strong phenotype that is qualitatively similar to the heterozygous phenotype for drug rescue that could be translatable for genetic forms of ASD.For instance, recently a clinical assessment of individuals with PMS found that a large minority suffer visual hyporeactivity, seeWalinga et al. 2022, European Journal of Medical Genetics.Therefore, our shank3 gene models may provide a phenotype that is similar to visually initiated hyporeactive symptoms present in individuals with shank3 variant PMS.We believe further research determining secondary effects of commonly prescribed drugs for any condition would benefit the knowledgebase of clinical researchers and doctors.
Response: Our figure legend title, "Figure4.LiCl does not impact lights-off activity or reactivity behaviors in wildtype and shank3ab mutants."Referred to the finding that no behavior changed when exposed to LiCL, wildtype or mutant.We have tried to address this issue by changing the figure legend title to be, "Figure4.LiCl does not impact lights-off VMR behaviors in wildtype and shank3ab mutants."Figure7.panel b and c, label WT and N-/+ and N -/-.In panel a, it appears the investigators assessed N-/-and N-/+.However, in the accompanying graphics, it appears only the N-/-is graphed.Is that correct?Is N-/+ a heterozygote?I do not see assessment of heterozygotes in the methods or anywhere else in the manuscript.Please include the methods in the methods section.