Larval zebrafish model for studying the effects of valproic acid on neurodevelopment: An approach towards modeling autism

Accepted Manuscript Larval zebrafish model for studying the effects of valproic acid on neurodevelopment: An approach towards modeling autism

Shubham Dwivedi, Raghavender Medishetti, Rita Rani, Aarti Sevilimedu, Pushkar Kulkarni, Perumal Yogeeswari PII: DOI: Reference:

S1056-8719(18)30619-1 https://doi.org/10.1016/j.vascn.2018.11.006 JPM 6548

To appear in:

Journal of Pharmacological and Toxicological Methods

Received date: Revised date: Accepted date:

6 April 2018 23 October 2018 21 November 2018

Please cite this article as: Shubham Dwivedi, Raghavender Medishetti, Rita Rani, Aarti Sevilimedu, Pushkar Kulkarni, Perumal Yogeeswari , Larval zebrafish model for studying the effects of valproic acid on neurodevelopment: An approach towards modeling autism. Jpm (2018), https://doi.org/10.1016/j.vascn.2018.11.006

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ACCEPTED MANUSCRIPT Larval zebrafish model for studying the effects of valproic acid on neurodevelopment: an approach towards modeling autism Shubham Dwivedi,a,# Raghavender Medishetti,b,# Rita Rani,b Aarti Sevilimedu,b Pushkar

Drug Discovery Research Laboratory, Department of Pharmacy, Birla Institute of Technology

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& Science-Pilani, Hyderabad Campus, Hyderabad-500078, India.

Dr. Reddy’s Institute of Life Sciences, University of Hyderabad Campus, Gachibowli,

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Hyderabad 500046, India. Equal Contribution

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#

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Kulkarni, a,b,* Perumal Yogeeswari.a,*

*Corresponding Authors:

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* Pushkar Kulkarni

Phone: +91-40-66571500, Fax: +91-40-66571581

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Email: [email protected], [email protected]

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* Perumal Yogeeswari

Phone: +91-4066303515, +91-40-66303823

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Email: [email protected], [email protected]

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ACCEPTED MANUSCRIPT LIST OF ABBREVIATIONS:

ADHD: Attention Deficit Hyperactivity Disorder ASD: Autism Spectrum Disorder

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dpf: days post fertilization

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DSM-5: Fifth edition of the Diagnostic and Statistical Manual of Mental Disorders

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E3: Embryonic media FDA: Food and Drug Administration

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FMRP: Fragile X mental retardation protein

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HDAC: Histone Deacetylase hpf: hours post fertilization

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IZ: Interaction Zone

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mGluR5: Metabotropic Glutamate Receptor 5

NRXN: Neurexin

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OFT: Open Field Test

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NLGN: Neuroligin

SHANK3: SH3 and multiple ankyrin repeat domains 3

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SZ: Social Zone

USFDA: United State Food and Drug Administration VPA: Valproic acid WHO: World Health Organisation

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ACCEPTED MANUSCRIPT ABSTRACT: Introduction: Autism Spectrum Disorder (ASD) is a complex neurodevelopmental disorder of early onset, characterized by impaired sociability, cognitive function and stereotypies. The etiology of ASD involves a multidimensional link between genetic, neurobiological and environmental factors. Since existing, comprehensive animal models for ASD are time

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consuming and laborious, the need for simple, quick approaches to study subsets of ASD-

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associated characteristics has always been in demand for better understanding of disease. The

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aim of the present study was to develop a cost and time effective zebrafish model with quantifiable parameters to facilitate mechanistic studies as well as high-throughput screening of

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new molecules for autism.

Methods: Zebrafish embryos were treated with valproic acid (75µM) beginning at 4-hours post

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fertilization to 5-days post fertilization. A series of behavioral tests (anxiety, inattentive behavior and circling behavior) and molecular studies were performed as surrogate parameters of ASD-

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like characteristic on the larvae at 7-dayspost fertilization for a quick screen. The study was followed by validation of model by screening positive control and negative control drugs. The

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social interaction test was performed on 21-days post fertilization to confirm that the surrogate phenotypes were indicative of social deficit (a core symptom of ASD).

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Results: The model showed a significant behavioral impairment (2-4fold difference) in valproic

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acid treated larvae compared to control larvae, which was further supported by alterations in select high-risk genes and proteins, implicated in human ASD. Reversal of behavioral impairments using standard drugs marketed for symptomatic treatment in ASD and no effect on

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behaviors when treated with paracetamol (negative control) signifies the role of model in preliminary drug screening. Conclusion: The model shows robust parameters to study behavior, molecular mechanism and drug screening approach in a single frame. Thus, we postulate that our 7-day larval model could be a useful preliminary screening tool to identify novel targets as well as potential drugs for autism and also can be applied to develop a high-throughput screening approach.

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ACCEPTED MANUSCRIPT KEYWORDS:

Autism,

Valproic

Acid,

Zebrafish

Larvae,

Behavioral

Assay,

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Neurodevelopmental Toxicity, Social Interaction

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ACCEPTED MANUSCRIPT INTRODUCTION: Autism is characterized by a diverse array of symptoms includingsocial deficits, repetitive behavior, attention deficit and language impairment.According to the fifth edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-5) autism spectrum disorder (ASD) is defined as, persistent deficits in social communication and interaction across several

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contexts, which are unveiled by deficits in social-emotional reciprocity, deficits in nonverbal

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communicative behaviors used for social interaction, or deficits in developing, maintaining and

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understanding relationships (Moussa, Srikrishnan, Blackwell, Dash, & Sibai, 2016). The prevalence of ASD has dramatically increased over the last few decades, affecting 1 out of 160 children worldwide (WHO, 2017), and is four times more likely to occur in males (Rynkiewicz

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et al., 2016). The etiology of autism is complex and multifactorial associated with impairment of several molecular pathways along with changes in genetic determinants and candidate genes

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such as SHANK3, NRXN and NGLN (Bourgeron, 2009; Jianling Chen, Yu, Fu, & Li, 2014;

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Stewart, Nguyen, Wong, Poudel, & Kalueff, 2014).

Children with autism may have one or more reported characteristic symptoms of autism

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(Dominick, Davis, Lainhart, Tager-Flusberg, & Folstein, 2007). Although a variety of autismlike symptoms can be mimicked in existing rodent models (Crawley, 2004; Roullet, Lai, &

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Foster, 2013), these models are not ideal for large scale drug discovery efforts (Toro et al., 2010). This is due to shortcomings such as costs, extensive labor, time-consuming maintenance,

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ethical considerations and lack in population approach (Belzung, Leman, Vourc’h, & Andres, 2005; Castro, Baronio, Perry, Riesgo, & Gottfried, 2016; Stewart et al., 2014). Since ASD is a

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complex disease with a number of varied underlying aetiologies, a true representative animal model that captures all of the phenotypes is unlikely to ever be available. Similarly, disease modifying therapies are also likely to be varied and combinatorial. This line of reasoning justifies the need to model subsets of ASD-associated characteristics in simpler models amenable to high throughput screening. Several lines of evidence have illustrated the utility of zebrafish as a model organism, which has gained significant attention in developmental biology, genetics, neurophysiology and biomedicine (Disorders, 2017; Gerlai, 2011; MacRae & Peterson, 2015; Spence, 2011).Given the previously established link between VPA treatment and ASD in rodents, we have extended the study to zebrafish to explore the possibility of a similar Page 5 of 31

ACCEPTED MANUSCRIPT connection. We have developed and validated a model for VPA induced neurodevelopmental injury which resemble certain ASD-associated phenotypes, and highlighted the potential of this model for drug screening. As the root of neurodevelopmental disorders like ASD lies within the in utero development period, ex vivo fertilization of zebrafish embryos with advantages like high fecundity, embryonic

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transparency, and rapid development makes it a choice for better understanding of disease.

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Recent reports suggest that zebrafish can serve as an excellent model to study ASD

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pathophysiology and genetics (Stewart et al., 2014; Tropepe & Sive, 2003), especially when valproic acid (VPA)is administered (Jiangfei Chen, Tian, Lei, Hou, & Roper, 2018). However, previous reports fail to provide method for use of zebrafish model in screening molecules, which

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highlights the novelty and exclusivity of our model. VPA, an anticonvulsant has been reported to be a potential risk factor in pathophysiology of autism during pregnancy (Chomiak, Turner, &

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Hu, 2013; Rodier, Ingram, Tisdale, & Croog, 1997; Roullet et al., 2013) and has been used in well recognized rodent models of ASD (Chomiak, Turner, and Hu 2013; Gottfried et al. 2013). It

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has been reported that VPA exposure in zebrafish results in phenotypic changes such as social deficit (Zimmermann, Gaspary, Leite, De Paula Cognato, & Bonan, 2015), altered neural

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progenitor cell proliferation (Y. Lee, Kim, Yun, & Lee, 2013) and deficit of serotinine neuronal differentiation (Jacob et al., 2014), which further supports the hypothesis that VPA could act as

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an inducing agent to modelselectautism associatedphenotypes in zebrafish.

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The aim of our study was to develop a cost and time efficient model with robust, quantifiable parameters associated with VPA induced neurodevelopmental toxicity and their relevance in

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autism, to facilitate mechanistic as well as preliminary screening studies. In the present study we have shown that VPA (75µM) treatment from 4-hours post fertilization (hpf) to 5-days post fertilization (dpf) induces ASD-like phenotypes. The series of behavior tests were validated to measure behavioral impairments (anxiety, inattentive attitude, circling, and social deficit), accompanied by altered expression of select genes and proteins implicated in ASD. All the parameters were studied on 7-dpf as a quick screen tool whereas social interaction was evaluated at 21-dpf. The parameters studied on 7-dpf were considered as surrogate tests for a quick 7-day screen. To validate them to be a true surrogate for the core symptom of ASD, social interaction was evaluated at 21-dpf. Moreover, the model was tested with standard, marketed drugs Page 6 of 31

ACCEPTED MANUSCRIPT (prescribed for symptomatic relief to ASD patients) along with negative control (paracetamol) on 7-dpf larvae. Thus we suggest that our 7-day larval model could be an approach towards preliminary screening to identify potentially new drugs as well as targets for autism.

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MATERIALS AND METHODS:

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Animal Ethics & Husbandry

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All zebrafish experiments were performed in accordance with the Institutional Bio-Safety Committee (DRILS/IBSC/2015/20); following ethical guidelines of Dr. Reddy’s Institute of Life

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Sciences, Hyderabad, India; as per the animal ethics laws of India. Mature wild caught zebrafish (Danio rerio) were maintained in circulating Millipore water with added essential minerals at

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28±1°C on a 12-hour light/dark cycle. All embryos were collected by natural spawning and were incubated at 28±1°C in embryonic (E3) media, (pH 7±0.5). The embryos of the same

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developmental stage were used for each experiment. All experiments were conducted at 28±1°C.

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The medium was changed according to prospects of the experiment. Drugs and Chemicals:

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Sodium valproate (Cat. No. P4543), fluoxetine (Cat. No. F132), aripiprazole (Cat. No. SML0935) and relcovaptan (Cat. No. S5701) were purchased from Sigma-Aldrich, St. Louis,

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MO, USA. Risperidone (Cat. No. R0087) and paracetamol (Cat. No. H0190) were purchased from Tokyo Chemical Industry (TCI) Co., Ltd.,Tokyo, Japan. These drugs were dissolved in the

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E3 medium. The cDNA synthesis kit (Cat. No. AB1453A) was purchased from Thermo Scientific, Waltham, Massachusetts, United States, and SYBR FAST qPCR kit (Cat. No. KK4601) was purchased from Kapa Biosystems India. All other chemicals used were of analytical grade. Standardization of VPA dose and exposure time: The standardization aimed to select the optimal dose as well as exposure time with no morphological defects and low mortality. The 4-hpf time-point was taken as a most suitable time for induction as reported by literature (Aluru, Deak, Jenny, & Hahn, 2013) and optimized in our Page 7 of 31

ACCEPTED MANUSCRIPT hands. At 4-hpf, embryos were exposed to different concentrations (3mM, 1mM, 300µM, 100µM, 75µM and 50µM) of VPA (Aluru et al., 2013; Herrmann, 1993; S. H. Lee, Kang, Lin, Lee, & Jin, 2013) and were observed every 24 h. The effect of VPA on zebrafish embryos/larvae is presented in supplementary data (Table S-1, Figure S-2). On the basis of observation, 75 µM was chosen as the highest non-toxic concentration, and various exposure paradigms (4-hpf to 3/5/7-dpf) were evaluated to find the best paradigm. Subsequently, to evaluate the merit of the

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disease model, the larval behavior in thigmotaxis/open field test was evaluated for highest non-

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toxic concentration. Further experiments were carried out at 75 µM VPA concentration with

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single exposure from 4-hpf to 5-dpf.

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Experimental Design:

The embryos were randomly divided into control and VPA treated group and were raised for 7-

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dpf. The larvae were treated with 75 µM VPA concentration with single exposure from 4-hpf to 5-dpf.The marketed drugs reported to rescue the symptoms were exposed from 5-dpf to 7-

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dpf.The behavioral tests were performed at 7-dpf between 10 am to 2 pm, followed by molecular studies. Larvae from each group were divided into two pools and were sacrificed (rapid freezing)

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for mRNA expression and protein studies. Few sets of larvae were raised up to 21-dpf to validate

Population Approach:

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ASD with social interaction test (Figure 1).

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We have used population approach for quantification of behaviors in this study. We have evaluated each behavioral parameter by quantifying the behavior in the group instead of

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evaluating single larvae. Details of quantification of each behavior are discussed below. The merits of population approach are discussed later in this manuscript. Behavioral parameters: All behavioral experiments were performed in a soundproof room under dim lights with restricted entry. The larvae were transferred to fresh E3 media 3-4 hours before the behavioral study and were taken to behavioral room 1 hour before the test. Different sets of larvae were used for each behavioral parameter. The tests were performed in quintuplicate with various sets of embryos to validate the reproducibility of the experiment.Analysis was done manually by an Page 8 of 31

ACCEPTED MANUSCRIPT observer blind to treatment. E3 media was replaced with fresh media after every trial. Statistical significance for all behavior parameters was ascertained using unpaired t-test. Outlines of behavioral methods have been presented in Figure 2. Diagrammatic representation for procedures and animations for principle of behavioral tests on 7-dpf has been provided in supplementary data (Figure S-3 and PowerPoint Presentation-S-4 respectively).

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Open Field Test (OFT): OFT utilizes the innate avoidance of novel open spaces to measure anxiety. The test was

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performed in a plastic petri dish filled with 3/4th volume of E3 media. Larvae (n=10) were placed in the centre of each dish at a time, and the video was recorded for 30 minutes. The number of

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larvae on edges of the plate (wall sticking behavior) was counted every minute to assess thigmotaxis behavior (Figure 2A). The results are reported as mean ± S.E.M. of the percentage

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of larvae on the edge of the plate.

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Inattentive Behavior Test:

This paradigm reflects the response of larvae to an aversive stimulus. The test was inspired by

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the available literature (Richendrfer & Créton, 2013) and necessary modifications were made to study the response of larvae towards aversive stimulus (Figure 2B). The plate was kept at the

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center of virtually divided two halves on the digital display. A PowerPoint presentation was used as an aversive stimulus for the larvae, starts with a blank white background (acclimatization) for

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30 minutes, followed by 30 minutes of a moving red bar (aversive stimulus) on the lower half of the plate. Video recording was done for both the phase of experiment. The number of larvae in

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the upper half of plate was counted after every 2 minutes. Inattentive attitude towards aversive stimulus was determined by the number of larvae which avoided the lower half, in VPA treated and control groups. The results were quantified by normalizing aversive stimulus period over acclimatization period as mentioned in the formula below, and are reported as mean ± S.E.M. of the percentage of larvae in upper half over acclimatization. % 𝐿𝑎𝑟𝑣𝑎𝑒 𝑖𝑛 𝑢𝑝𝑝𝑒𝑟 ℎ𝑎𝑙𝑓 𝑜𝑣𝑒𝑟 𝑎𝑐𝑐𝑙𝑖𝑚𝑎𝑡𝑖𝑧𝑎𝑡𝑖𝑜𝑛 =(

𝐴𝑣𝑒𝑟𝑠𝑖𝑣𝑒 𝑠𝑡𝑖𝑚𝑢𝑙𝑢𝑠 − 𝐴𝑐𝑐𝑙𝑖𝑚𝑎𝑡𝑖𝑧𝑎𝑡𝑖𝑜𝑛 ) ∗ 100 𝐴𝑐𝑐𝑙𝑖𝑚𝑎𝑡𝑖𝑧𝑎𝑡𝑖𝑜𝑛 Page 9 of 31

ACCEPTED MANUSCRIPT Circling Behavior Test: Circling is a repetitive swimming in a circular motion (usually seen during seizures and neurological impairments). The test was performed in a plastic petri dish filled 3/4th with E3 media. The set of 20 larvae (to make the plate crowded) were placed in the dish at a time, and the video was recorded for 30 minutes (Figure 2C). The number of circling movements were

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counted for the group of larvae (n=20) over a 30 minutes duration, and the results were reported

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as mean ± S.E.M. of a number of circling moments in 30 minutes to assess repetitive stereotypic

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behavior.

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Gene Expression Studies:

Total RNA was isolated from the fresh pool of larvae (n=50) tissue using the TRIzol method (Invitrogen, CA) following manufacturer’s instructions. The quality of RNA was confirmed by

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running RNA samples on 1% agarose gel, whereas the quantity of RNA (µg/ml) was analyzed using Eppendorf Bio Spectrometer (Hamburg, Germany). The RNA (1 µg) was converted in to

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cDNA by using Verso cDNA synthesis kit (Thermo Scientific) as per the manufacturer’s instructions. Gene expression was quantitatively determined by Real-Time PCR using Kappa

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SYBR® Green PCR Kit (Kappa Bioscience) and the CFX ConnectReal-Time PCR System (Biorad, USA) according to the manufacturer´s instructions. The primers (IDT India) used for

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amplification are mentioned in supplementary data (Table S-5). The primers were validated for linearity and specificity of amplification before the experiment. All reactions were performed in

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triplicate. The results are expressed relative to a β-actin, which is used as an internal control and are reported as mean ± S.E.M. Statistical significance was ascertained using two-way ANOVA

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followed by Bonferroni correction. Protein Level Studies: For protein expression study using Western Blot, the heads were dissected from ten 7-days old zebrafish larvae, pooled and homogenized in 20 μl SDS-sample buffer (2 μl/larvae) under liquid nitrogen. Samples were then incubated at 95 °C for 5-minutes followed by centrifugation at 10,000g for 10-minutes at 4°C. Whole protein lysate was resolved by12% SDA-PAGE. Western blot was carried out as per standard protocol to detect mGluR5 and FMRP, monoclonal antiPage 10 of 31

ACCEPTED MANUSCRIPT mGluR5 antibody (1:2000; Cat. No. GTX125996, GeneTex, Inc. (USA); anti-FMRP antibody (1:1000; Cat. No. AB5675, Merck, New Jersey, United States).The membrane was developed by ECL (enhanced chemiluminescence reagent), and the signals were captured using X-ray film, further analyzedby densitometry. The results are expressed relative to a β-tubulin, which is used as an internal control and are reported as mean ± S.E.M. Statistical significance was ascertained

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Validation of Model with Positive and Negative Control Drugs:

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using two-way ANOVA followed by Bonferroni correction.

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The recognition of any disease model is based on its utility as a screening tool . Also for validation of the model, available therapeutics should be able to rescue the disease like condition

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either partially or completely. However, as no drugs have been approved for overall treatment of autism and only symptomatic treatment is available, the validation of this model is a major

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challenge. Nevertheless, the positive and negative control drugs tested for validation and the rationale for their use is mentioned in the following statements. Aripiprazole and risperidone are

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the only drugs approved by United State Food and Drug Administration (US FDA), specifically for irritability associated with autism (US FDA 2017, US FDA 2018, Kirino, 2014). Fluoxetine

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is a drug frequently used in clinics for autism (Mouti et al., 2014; Taylor, 2016). Reclovaptan is a vasopressin receptor antagonist selective to V1a subtype (Decaux, Soupart, & Vassart, 2008); we

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selected it as a positive control due the fact that its mechanism is identical to that of balovaptan which has recently been provided breakthrough therapy designation for autism by US FDA

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(DeFrancesco, L. 2018). Balovaptan is presently in Phase III clinical trials and was inaccessible (not commercially available in Hyderabad, India at this point in time)to us, hence we tested

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reclovaptan. Paracetamol, was screened as negative control as it may provoke autism when taken during pregnancy or after birth (Good, 2009). Therefore, four positive control drugs (two approved for symptomatic treatment, one clinically used and one identical to a drug having clinical proof-of-concept) and one negative control were used to validate this model. The drugs were tested at 3 different doses (described in result section), below the highest nontoxic concentration. The larvae were exposed (single exposure) to E3 media containing drug from 5-dpf to 7-dpf. The media was changed 3 hours before behavioral assays. Results are

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ACCEPTED MANUSCRIPT reported as mean ± S.E.M. and statistical significance was ascertained using one-way ANOVA followed by Dunnett post hoc test. Social Interaction Test: Social interaction test was performed to validate one of the core symptoms linked with autism in

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this model. Since it is hard to study social behavior in 7-dpf larvae, we have designed a test to

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study the social interaction of larval zebrafish at 21-dpf, based on the previously reported literature (Dreosti, Lopes, Kampff, & Wilson, 2015). Certain modifications were made to

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perceive the social behavior in our laboratory. Experiments were performed in a custom-built behavioral setup, divided into 5 chambers (1 central, 2 test, 2 blank), central and blank chamber

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were visible from test chamber so that the action of fish towards different chambers can be visualized. The E3 media was filled to half of the height of the plate. Ten larvae were first

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transferred to the central chamber followed by transfer of control and VPA treated larvae, one larva in one test chamber (near the glass window of the central chamber) simultaneously. Video

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recording was done for 7 minutes with initial1 minute for acclimatizationfollowed by 6 minutes for analysis. The test chamber was virtually divided into social and interaction zone for analysis

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(Figure 2D). The percentage of time spent by larvae in the interaction and the social zone was observed and the results were analyzed as percentage time spent by larvae in social and

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interaction zone and are reported as mean ± S.E.M.Diagrammatic representation for procedures

RESULTS:

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of social interaction test is provided in supplementary Figure S-6.

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Effect of VPA on Behavioral Parameters Raw data for all behavioral tests have been provided in supplementary data (Excel S-7). Thigmotaxis Behavior: In theOFT, a 30 minutes video analysis revealed that the larvae settles to the wall over a period of time hence the wall sticking behavior was not due to immobility. (Supplementary Figure S-8A). Additionally, the data from the last five minutes of recording looks similar to the data compiled over the 30 minute window, (supplementary Figure S-8B), thus to make this test quick and robust we strongly recommend considering last 5 minutes data for analysis of thigmotaxis. A significantly high (p<0.001) wall sticking behavior was observed Page 12 of 31

ACCEPTED MANUSCRIPT with VPA treated larvae in comparison to control (Figure 3A), which represents poor exploration and ultimately symbolizes anxiety. A representative video is included in the supplementary data(S-9). Inattentive behavior: During acclimatization (30 minutes), control as well as VPA treated larvae moved randomly throughout the plate. During aversive stimulus phase, we observed a significant

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(p<0.001) shift of control larvae to the upper half of plate, away from the moving red bar. This

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intent to avoid the “aversive” stimulus was not present in the VPA treated larvae, which

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continued to occupy both halves of the plate (Figure 3B). We believe that this inability to detect and/or comprehend the presence of the aversive stimulus (unconcerned/inattentive attitude) in the VPA treated larvae, maybe reflective of a cognition deficit.Figure S-10summarizes the

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activity of larvae over the period of complete test and a representative video is included in the

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supplementary data (Video S-11).

Circling Behavior: Significantly higher circling (P<0.001) was observed in larvae treated with

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VPA as compared to control (Figure 3C). The number of circling events observed was four folds higher in the VPA treated larvae as compared to control larvae. Arepresentative (VideoS-12)is

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provided as part of the supplementary data for better understanding of circling behavior.

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Effect of VPA on Gene Expression: We selected 3 potential genes (SHANK3a, NRXN 1 and NGLN 3) which have been reported to

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display altered expression in autism (https://sfari.org/resources/sfari-gene). The percentage homology (Table S-13), gene scoring in relevance to autism (ExcelS-14) and basis of selection of genes and protein (Word S-15) have been provided as supplementary data. Our findings suggest a significant reduction in expression level of all three genes, SHANK3a (P<0.001), NRXN 1 (P<0.01) and NLGN 3 (P<0.05) in larvae exposed to VPA as compared to matched controls (Figure 3D).While changes in the expression of just three candidate genes is insufficient to reveal the molecular basis of the VPA induced phenotypes, these changes are consistent with what is observed in humans with ASD (Betancur, Sakurai, & Buxbaum, 2009)and indicate the potential for overlapping pathways for VPA induced and ASD related molecular changes.VPA is Page 13 of 31

ACCEPTED MANUSCRIPT a known HDAC inhibitor, and is expected to result in broad changes in gene expression patterns, which will be the subject of future studies in this model. Effect of VPA on Protein Level: FXS is a major monogenic cause of ASD. In FXS, the level of FMRP is decreased due to

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silencing of the gene, leading to an upregulation of the metabotropic glutamate receptor

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(mGluR5) and consequent changes in the signaling cascades downstream (Lohith et al., 2013). It is believed that the ASD-like phenotypes of FXS are rooted primarily in these changes in FMRP

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and mGluR5 proteins levels. So we measured these proteins in our model to determine if VPA worked through a similar pathway. In the VPA treated 7-dpf larvae, there was a decrease

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(insignificant) in FMRP level, however, a significant increase in mGluR5 protein level was observed (Figure 3E) as compared to control, when quantified using a standard western blot

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experiment. The representative western blot images are presented in figure 3F and figure 3G. This result suggests that in the VPA model, the increased mGluR5 level and accompanying

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changes in the glutamate signaling cascades could contribute significantly to the behavioral phenotypes observed, which in turn can be a source for behavioral impairment as suggested in

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autism (Betancur et al., 2009; Mehta, Gandal, & Siegel, 2011).

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Effect of Positive and Negative Control Drugs on VPA Induced Behavioral Despair: The pharmacological interventions explored as positive control were able to rescue one or more

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behavioral parameters observed in the model. Aripiprazole shows a significant rescue of thigmotaxis behavior at 0.5 µM (p<0.05) and 1 µM (p<0.001), inattentive and circling behavior

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at all explored concentration i.e. 0.25 µM (p<0.001), 0.5 µM (p<0.05) and 1 µM (p<0.01 inattentive, p<0.001 circling) when compared to VPA control group. Risperidone in comparison to VPA control group, significantly reverses the thigmotaxis at 1µM (p<0.05) and3 µM (p<0.001), inattentive behavior at 3 µM(p<0.001),whereas, circling was abrogated at all tested concentration 0.3 µM (p<0.05), 1 µM (p<0.001) and 3 µM(p<0.001) of risperidone. Fluoxetine shows significant effect in rescue of thigmotaxis at 0.5 µM (p<0.01), inattentiveness at 1 µM (p<0.01), however, fluoxetine significantly reduces circling behavior at 0.5 µM (p<0.05) and 5 µM (p<0.05). Relcovaptan reverses thigmotaxis and circling behavior in a dose dependent manner with significant effect for thigmotaxis at 3 µM (p<0.001) and 10 µM (p<0.001) and Page 14 of 31

ACCEPTED MANUSCRIPT circling at 1 µM (p<0.05)3 µM (p<0.001) and 10 µM (p<0.001). Relcovaptan also rescues inattentive behavior at 1 µM. Paracetamol serving as negative control shows no rescue for the VPA induced behavioral despair observed in the model. Reversal of behavioral phenotypes with available symptomatic treatment encourages the idea of using this model in screening candidate molecules.

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Effect of VPA on Social Interaction:

Impaired social interaction is one of the core symptoms of autism. We found that control larvae

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spent a significant percentage of time in social (p<0.01) and interaction (p<0.01) zone as compared to VPA treated larvae. Although the VPA treated larvae spent time in the social zone

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(significantly less as compared to control), they rarely entered the interaction zone. This suggests that the VPA treated larvae avoided interaction despite visualizing the conspecifics.

Those

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larvae that spent over 25% time in the social zone and interaction zone were labelled ‘socially active larvae’ and ‘interaction active larvae’ respectively. The frequency of socially active larvae

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(p<0.05), and interaction active larvae (p<0.01) were significantly higher in the control group as compared to the VPA treated group (Figure 4). Thus from social interaction test, it can be

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concluded that VPA treated larvae show a significant social deficit in comparison to control larvae. Raw data and representative video for social interaction have been provided in

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DISCUSSION:

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supplementary information (Excel S-7) and Video S-16 respectively.

In the present study, we report a novel, robust, and economical model in zebrafish, to study the effect of VPA in neurodevelopment and its relevance to ASD. The techniques used in this model are in-house and can be performed with basic facilities thus making it cost effective. Here, for the first time we report the population approach in zebrafish larvae, as it has been hypothesised that individual organisms (zebrafish or mice or humans) suffering from a complex disorder such as autism may not represent all the symptoms coupled with disease. Moreover, population approach also offers simple and rapid quantitative approach for each of the symptom being evaluated in this study. Despite of several advantages of population approach, it provide a Page 15 of 31

ACCEPTED MANUSCRIPT generalized idea about the impact of treatment in any study and fails to explain the effect of treatment on individual. However, we believe that population approach could help in medium to high throughput screening at early stages of drug discovery. We have identified an optimal non-toxic concentration of VPA which produces robust phenotypes with no signs of visible morphological defects and less mortality. Visible behavioral

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changes such as (1) anxiety (2) inattentive behavior towards the aversive stimulus (3) high

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circling behavior and (4) social deficit are noted in VPA treated larvae when compared to the

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control group. Interestingly, we also found changes in gene expression and protein levels involved in neurodevelopment and synapse formation, which also plays a role in development and progression of disease such as autism. We scrutinized the model with symptomatic therapy

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used in autism to confirm the reversal of behavioral deficit. Most importantly, this model also has several advantages over rodent models: i) small size and less maintenance ii) high fecundity,

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iii) rapid and transparent ex utero embryonic development, v) genomic similarities with human

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and vi) population approach (Ali, Champagne, Spaink, & Richardson, 2011; Best et al., 2008). Several previous studies corroborated that VPA has definitive role in mimicking ASD like

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feature in zebrafish (Meshalkina et al., 2018). It has been reported that VPA exposure at early embryonic stage does not affect the survival, morphology, and hatching rate, but may induce

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some alterations in locomotor activity and can produce anxiety at 6-dpf, with no reports of behavioral abnormalities in adults (Zimmermann et al., 2015). Literature suggest that VPA

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exposure can provoke ASD-like physical characteristics and behavioral impairments like hyperactivity and social deficit in zebrafish (Jiangfei Chen et al., 2018) and is also reported to

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modulate expression and splicing of SHANK3 in zebrafish (Liu et al., 2016).Therefore, our experimental data together with the afore mentioned facts further provides a strong correlation for implicating the use of zebrafish model as an alternative and newer approach for mimicking the ASD like phenotypes and for investigating its underlying pathophysiology. In our model, higher rate of thigmotaxis has been assumed to be linked with either exploration or predator avoidance in fish and anxiety-like behavior in mammals (Bhinder & Tierney, 2012). In general, any species show avoidance to unpleasant stimulus, considered as passive avoidance. The passive avoidance learning has been previously emulated in zebrafish (Ahmed, Seguin, & Page 16 of 31

ACCEPTED MANUSCRIPT Gerlai, 2012). In inattentive behavior test, unlike control larvae, which shows passive avoidance towards aversive stimulus we have observed the unconcerned behavior of VPA treated larvae to the aversive stimulus. Circling, a repetitive swimming in a circular motion is a typical representative of the irritability (Lewis, Tanimura, Lee, & Bodfish, 2007; Stewart et al., 2014). The irritability and repetitive behavior in rodents includes self-grooming, head bobbing and marble burying, and is reported as increased stereotypic behavior in VPA-induced or genetic

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rodent model of autism (Mcfarlane, Kusek, & Yang, 2008; Schneider & Przewlocki, 2004). In

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regard to social interaction, 7-dpf larvae have been reported to show poor social preference,

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whereas most of the 3 weeks old larvae show a strong social preference (Dreosti et al., 2015). Social preference is vision dependent as it requires visualizing a fish of a similar age/size. The

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larval zebrafish do not express the overt shoaling and schooling behaviors like adults, however, they prefer to approach and remain near conspecifics. The entries of VPA treated zebrafish in

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the social zone with rare entries into interaction zone justifies that the VPA treated fish are able to visualize the conspecifics and have motor activity but are not willing to interact or come

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closure to conspecifics. In each of the behavioral paradigms tested, the VPA treated larvae has shown significant behavioral abnormalities.

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A doubt can be raised suggesting that the behaviors of thigmotaxis and inattentiveness could be attributable to visual and locomotor dysfunction, however, the videos (Videos S-9 & S-11) and

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the pattern of data in supplementary information (Figures S-8 & S-10) will convince the readers that these are indeed behaviors of anxiety and inattentiveness. Furthermore, the social interaction

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test also confirms the visual and locomotor abilities of the larvae.

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We have used four positive control drugs; aripiprazole, risperidone, fluoxetine and relcovaptan and one negative control paracetamol. The rationale of using the various controls has been descried in the methods sections. Despite the challange of validation due to lack of approved therapies, the results from our study demonstrate that, positive control drugs used in the study reverse VPA-induced abnormal behaviors and the negative control did not, thus providing the initial validation of this model. Evidence from previous studies suggests that VPA exposure leads to behavioral despair, but the mechanism behind is not well understood yet (Roullet et al., 2013; Zimmermann et al., 2016). Page 17 of 31

ACCEPTED MANUSCRIPT Histone deacetylase (HDAC) inhibition plays a critical role in VPA induced congenital disabilities (Fukuchi et al., 2009; Phiel et al., 2001). Prenatal HDAC inhibition delays neuronal maturation by alterations in the expression of NRXN, NLGN and SHANK3 (Kawanai et al., 2016)among others. A number of studies in rodent have theorized the possible link between select genes and the observed behavioral phenotypes. For example: increased grooming behavior was observed in NRXN1 deficient mice (Jianling Chen et al., 2014); NLGN3 is associated with

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social interaction deficits, increased spatial memory and an electrophysiological phenotype. The

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KO of NLGN3 leads to reduced ultrasound vocalization and deficits in social novelty preference

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(Radyushkin & Hammerschmidt, 2009). Haploinsufficiency of SHANK3 relates to changes in cellular components linked with social cognition (Bozdagi et al., 2010) Therefore, our

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observation of a similar change in expression pattern for these three candidate genes provides a probable link between the action of VPA at the molecular level and the resultant phenotypes we

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observe. The upregulation of mGluR5 in the present study are in tune with the previous reports which implicate the glutamate signalling cascade in neurodevelopmental defects

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(Jacquemont et al., 2011; Mehta et al., 2011). Hence, with the present study we have tried to draw lines between effect of VPA induction in early embryonic age on synaptic proteins and

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behavioral changes (Figure 5).

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Conclusion

Briefly, the root cause of ASD lies deep in prenatal fate (Moussa et al., 2016) and this stage is

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uniquely accessible in the zebrafish model due to the ex vivo embryonic development. Our model uses VPA for induction of behavior despairs along with molecular level changes which

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are also observed in ASD. To the best of our knowledge, this study is the first of its kind to report a comprehensive model with detailed paradigm for disease induction, behavioral characterization, selected mechanistic and predictive approaches in a single frame. Therefore, we propose the use of 7-days old larval zebrafish for preliminary screening of drugs against VPA induced neurodevelopmental toxicity, which can be further extended to 21-days for testing of lead molecules on the social deficit. The methods described here could potentially be utilized to develop a high-throughput screening approach for screening molecules for ASD. However, further studies, such as multi-laboratory large scale validation and whole genomic /proteomic analysis are required for comprehensive validation of this model. Page 18 of 31

ACCEPTED MANUSCRIPT CONFLICT OF INTEREST STATEMENT: The authors have no conflict of interest (commercial or otherwise) to disclose. FUNDING & ACKNOWLEDGEMENTS: Authors are grateful to Birla Institute of Technology and Science (BITS) – Pilani, Hyderabad

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Campus (India) and Dr. Reddy’s Institute of Life Sciences (India) for infrastructural facilities,

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research resources, and, PhD registration to the lead author. This work was supported by internal

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funding from the affiliations.

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Behavioural Parameters

Open Field Test

Doses)

for each

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Groups (Controls &

Replicates (n)

% Larvae Sticking

behavior

M

to Wall

Aripiprazole (Positive Control)

Test % Larvae in upper half over acclimatization (Mean ± S.E.M.)

Circling

Number of Circling Moments (Mean ± S.E.M.)

6

39.33 ± 3.75***

45.06 ±3.83***

19.33 ± 1.48***

6

74.50 ± 3.05

9.30 ± 3.71

68.67 ± 6.21

6

64.93 ± 10.91

38.20 ± 4.86***

45.67 ± 2.87***

6

42.87 ± 6.67*

24.12 ± 2.37*

53.67 ± 1.84*

6

27.55 ± 6.51***

28.54 ± 3.67**

30.33 ± 1.80***

Vehicle Control

6

36.03 ± 2.92***

37.77 ± 4.98***

19.67 ± 2.79***

VPA Control

6

62.37 ± 3.03

12.81 ± 2.56

85.67 ± 6.21

0.3 µM

6

61.94 ± 2.72

17.85 ± 1.32

63.67 ± 4.07*

0.25 µM

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0.5 µM

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VPA Control

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Vehicle Control

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(Mean ± S.E.M.)

Inattentive Behavior

1 µM

Risperidone (Positive Control)

Page 19 of 31

1 µM

6

52.04 ± 1.65*

16.19 ± 1.80

45.67 ± 4.51***

3 µM

6

45.38 ± 3.31***

34.92 ± 5.24**

41.33 ± 5.35***

Vehicle Control

6

39.67 ± 3.59***

36.23 ± 4.73**

18.67 ± 2.16***

VPA Control

6

62.67 ± 3.89

2.71 ± 6.06

58.50 ± 4.10

0.5 µM

6

45.67 ± 2.43**

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ACCEPTED MANUSCRIPT

19.61 ± 1.92

35.67 ± 3.46*

1 µM

6

70.33 ± 3.40

39.27 ± 8.91**

42.67 ± 7.71

5 µM

6

62.67 ± 3.37

Vehicle Control

6

30.36 ± 3.18***

VPA Control

6

1 µM

6

3 µM

6

10 µM

2.96 ± 10.57

33.50 ± 7.94*

37.63 ± 2.70***

16.67 ± 4.20***

55.16 ± 1.95

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6.26 ± 1.55

96.33 ± 10.91

52.90 ± 1.01

27.5 ± 10.03*

71.33 ± 7.25*

44.09 ± 1.30***

13.47 ± 0.81

35 ± 2.76***

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Fluoxetine (Positive Control)

38.60 ± 0.48***

12.24 ± 1.13

27.67 ± 1.12***

6

37.53 ± 1.49***

38.75 ± 4.78***

22.33 ± 1.48***

6

61.83 ± 4.77

12.37 ± 1.12

82.00 ± 6.34

6

66.34 ± 3.20

5.68 ± 2.38

76 ± 5.53

30 µM

6

63.98 ± 1.03

7.95 ± 5.66

73.33 ± 3.31

100 µM

6

65.59 ± 0.67

11.21 ± 2.41

82.67 ± 7.68

6

VPA Control

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10 µM

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Vehicle Control

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Paracetamol (Negative Control)

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Relcovaptan (Positive Control)

Table 1: Effect of Positive Control and Negative Control Drugs on VPA Induced Behavioral Despair. The effect of positive and negative control drugs on behavioral parameters was observed on 7 th days post fertilization. Effect of each drug was compared with the vehicle control and VPA control of same experiment. Results are represented as mean ± S.E.M. (n=6 replicates (2 Experiments X 3 plates/experiment)) and statistical significance was analysed using one-way ANOVA followed by Dunnett post hoc test in GraphPad Prism® software. p<0.05 was Page 20 of 31

ACCEPTED MANUSCRIPT considered to be significant. The vehicle control and test groups were compared against VPA control group, where

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*p<0.05, **p<0.01 and ***p<0.001.

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neurodevelopmental causes of autism? Genes, Brain and Behavior, 2(5), 268–281.

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https://doi.org/10.1007/s10803-006-0117-y US FDA, (2017). ABILIFY® Label Sheet.

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https://www.accessdata.fda.gov/drugsatfda_docs/label/2017/021436s042,021713s033,0217 29s025,021866s027lbl.pdf. Accessed at 21 October 2018 US FDA, (2018). RISPERDAL® Label Sheet. https://www.accessdata.fda.gov/drugsatfda_docs/label/2018/020272s080,020588s068,021444s05 4lbl.pdf. Accessed 21 October 2018 WHO (2017). Home/News/Fact sheets/Detail/Autism spectrum disorders http://www.who.int/news-room/fact-sheets/detail/autism-spectrum-disorders. Accessed 21 October 2018. Page 27 of 31

ACCEPTED MANUSCRIPT Zimmermann, F. F., Gaspary, K. V., Leite, C. E., De Paula Cognato, G., & Bonan, C. D. (2015). Embryological exposure to valproic acid induces social interaction deficits in zebrafish (Danio rerio): A developmental behavior analysis. Neurotoxicology and Teratology, 52, 36– 41. https://doi.org/10.1016/j.ntt.2015.10.002 Zimmermann, F. F., Gaspary, K. V., Siebel, A. M., Leite, C. E., Kist, L. W., Bogo, M. R., &

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Bonan, C. D. (2016). Analysis of Extracellular Nucleotide Metabolism in Adult Zebrafish

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After Embryological Exposure to Valproic Acid. Molecular Neurobiology, 1–12.

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https://doi.org/10.1007/s12035-016-9917-z

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ACCEPTED MANUSCRIPT F IGURE LEGENDS Figure 1: Experimental design of proposed zebrafish larvae model Embryos were exposed to VPA at a single dose of 75 µM from 4-hpf to 5-dpf. Fluoxetine and aripiprazole were administered from 5-dpf to 7-dpf. Behavioral and molecular studies were

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conducted on 7-dpf. To confirm ASD-like phenotypes social interaction was performed at 21-dpf

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Figure 2: Pictographic representation of behavioral assessment chambers used in model

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A) Open Field Test: The test was performed in a plastic petri dish with dimensions 90 mm diameter and 15 mm height. The number of embryos used per plate n=10.

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B) Inattentive Behavior Test: The test assembly consist of plastic tray measuring 200 mm x 150 mm. In this tray 150 ml agarose gel (2% in E3 media) was poured onto which 5

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depressions (lanes) were made using glass slides, with final dimension of each lane as 75 mm x 25 mm x 5mm, the gel was allowed to cool. The plate was positioned over the centre of

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of embryos used per lane n=10.

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digital screen and the red bar was allowed to move on the lower half of the plate. The number

C) Circling Behavior Test: The test was performed in a plastic petri dish with dimensions 90

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mm diameter 15 mm height. The number of embryos used per plate n=20. D) Social Interaction Test: The test assembly was a plastic tray which measures 125 mm X 85

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mm X 15 mm. The tray was filled up to full capacity with warm agarose gel soluti on (2% in E3 media) and allowed to cool. The assembly was designed by cutting and removing

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excessive gel and was further divided using glass partition into 5 chambers with specific dimensions. One central chamber: 20 mm X 20 mm, two test chamber: 65 mm X 20 mm, two blank chambers: 40mm X 20 mm. The test chamber was virtually divided into social and interaction zone for analysis. Interaction zone: 5 mm X 20 mm (1/4th of total width) and Social zone: 13 mm X 20 mm (2/3rd of total width) from central chamber glass partition. The embryos were analysed individually in each test chamber.

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ACCEPTED MANUSCRIPT Figure 3: Behavioral and molecular changes on zebrafish larvae with 75 µM VPA at 7-dpf. A) Open field test: analysis of wall sticking behavior (n=15 replicates (5 experiments X 3 plates/experiment); 10 larvae/plate (replicate). B) Inattentive behavior: The response to aversive stimulus over acclimatization was assessed (n= 25 replicates (5 experiments X 5 plates/experiment); 10 larvae/plate (replicate). C) Circling behavior: Number of circling were

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counted for 30 minutes (n= 25 replicates (5 experiments X 5 plates/experiment); 20 larvae/plate

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(replicate). D) The expression level of high-risk genes (SHANK3a, NRXN 1 and NGLN 3) for autism from 3 replication of study (n = 3 X 50). E) Relative protein expression (mGluR5 and

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FMRP) in VPA treated larvae over control (n=3 X 10). F) Representative image of mGluR5 protein expression as measured by Western blot in zebrafish larvae. G) Representative image of

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FMRP protein as measured by Western blot in zebrafish larvae. All results are reported as mean ± S.E.M. and statistical significance was ascertained using un-paired t-test for behavioral

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parameter and two-way ANOVA followed by Bonferronitest for gene and protein expression in GraphPad Prism® software, where ***p<0.001, **p<0.01 and *p<0.05 as compared to control

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group.

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Table 1: Effect of Positive Control and Negative Control Drugs on VPA Induced Behavioral Despair:

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The effect of positive and negative control drugs on behavioral parameters was observed on 7th days post fertilization. Effect of each drug was compared with the vehicle control and VPA

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control of same experiment. Results are represented as mean ± S.E.M. (n=6 replicates (2 Experiments X 3 plates/experiment)) and statistical significance was analysed using one-way

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ANOVA followed by Dunnett post hoc test in GraphPad Prism® software. p<0.05 was considered to be significant. The vehicle control and test groups were compared against VPA control group, where *p<0.05, **p<0.01 and ***p<0.001. Figure 4: Social interaction pattern changes in zebrafish larvae due to 75 µM VPA at 21dpf: A) Percentage time spent by larvae in the social zone (n=24). B) Percentage time spent by larvae in interaction zone (n=24). C) Percentage frequency of larvae spending more than 25% of total time in the social zone (socially active larvae) (n=24). D) Percentage frequency of larvae Page 30 of 31

ACCEPTED MANUSCRIPT spending more than 25% of total time in interaction zone (interaction active larvae) (n=24). Values in above mentioned graphs are expressed as mean ± SEM and the statistical significance was ascertain using unpaired t-test, where **p<0.01 and *p<0.05 as compared to control group.

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Figure 5: Pathway of autistic behavior linked with the effect of VPA on Synaptic proteins.

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The figure shows the connection between VPA, HDAC1 and synaptic proteins thought to be involved in autism. The proteins of interest which were assessed in this study are color coded as

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follows: red indicates downregulation, green indicates up-regulation in protein levels or gene expression. HDAC1 is an important target of VPA in the pathogenesis of congenital disabilities,

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which further regulates various genetic and epigenetic factors involved in neuronal development. VPA may have direct or indirect (HDAC1 inhibition) effect on synaptic proteins and receptors.

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The alteration in gene expression and protein levels leads to defects in synaptogenesis and early neural development, consequently leading to Autism behaviors like social interaction, attention

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deficit, anxiety, impaired cognition, repetitive stereotypic behavior etc.

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