Inhibition of Lrrk2 reduces ethanol preference in a model of acute exposure in zebrafish

Journal Pre-proof Inhibition of Lrrk2 reduces ethanol preference in a model of acute exposure in zebrafish

Isadora Marques Paiva, Luana Martins de Carvalho, Isabela Martins Di Chiaccio, Isadora de Lima Assis, Elena Naranjo Sánchez, Manuel Bernabé Garcia, Felipe Norberto Alves Ferreira, Maria Luisa Cayuela, Luis David Solis Murgas, Ana Lúcia Brunialti Godard PII:

S0278-5846(19)30628-1

DOI:

https://doi.org/10.1016/j.pnpbp.2020.109885

Reference:

PNP 109885

To appear in:

Progress in Neuropsychopharmacology & Biological Psychiatry

Received date:

26 July 2019

Revised date:

28 December 2019

Accepted date:

3 February 2020

Please cite this article as: I.M. Paiva, L.M. de Carvalho, I.M. Di Chiaccio, et al., Inhibition of Lrrk2 reduces ethanol preference in a model of acute exposure in zebrafish, Progress in Neuropsychopharmacology & Biological Psychiatry(2020), https://doi.org/10.1016/ j.pnpbp.2020.109885

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

© 2020 Published by Elsevier.

Journal Pre-proof Inhibition of Lrrk2 reduces ethanol preference in a model of acute exposure in zebrafish Isadora Marques Paiva1 , Luana Martins de Carvalho1 , Isabela Martins Di Chiaccio2 , Isadora de Lima Assis2 , Elena Naranjo Sánchez3 , Manuel Bernabé Garcia3 , Felipe Norberto Alves Ferreira4 , Maria Luisa Cayuela3 , Luis David Solis Murgas2 , Ana Lúcia Brunialti Godard1 * 1

Laboratório de Genética Animal e Humana, Departamento de Genética, Ecologia e

Evolução, Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, Brazil 2

Biotério Central, Departamento de Medicina Veterinária, Universidade Federal de Lavras

Aging Cancer and Telomerase Laboratory, Instituto Murciano de Investigación Biosanitaria

oo

3

f

(UFLA), Lavras, Brazil.

Virgen de la Arrixaca, Murcia, Spain.

Laboratório de Nutrição Animal, Departamento de Medicina Veterinária, Universidade

pr

4

e-

Federal de Minas Gerais (UFMG), Belo Horizonte, Brazil

Pr

*Corresponding author: Ana Lúcia Brunialti Godard. Laboratório de Genética Animal e Humana, Departamento de de Genética, Ecologia e Evolução, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais. Avenida Antônio Carlos, 6627 – Campus

al

Pampulha – Belo Horizonte – MG – CEP 31270-901 – Brazil.

Jo u

Abstract

rn

E-mail: [email protected] Phone +5531-34092594.

Due to its multifactorial and yet to be fully understood origin, ethanol addiction is a field that still requires studies for the elucidation of novel genes and pathways that potentially influence the establishment and maintenance of addiction-like phenotypes. In this context, the present study aimed to evaluate the role of the LRRK2 pathway in the modulation of ethanol preference behavior in Zebrafish (Danio rerio). Using the behavioral Conditioned Place Preference (CPP) paradigm, we accessed the preference of animals for ethanol. Next, we evaluated the transcriptional regulation of the gene lrrk2 and the receptors drd1, drd2, grin1a, gria2a, and gabbr1b in the zebrafish brain. Additionally, we used a selective inhibitor of Lrrk2 (GNE-0877) to assess the role of this gene in the preference behavior. Our results revealed four distinct ethanol preference phenotypes (Light, Heavy, Negative Reinforcement, and Inflexible), each showing different transcriptional regulation patterns of the drd1, drd2, 1

Journal Pre-proof grin1a, gria2a, and gabbr1b receptors. We showed that the lrrk2 gene was hyperregulated only in the brains of the animals with the Inflexible phenotype. Most importantly, we showed, for the first time in the context of preference for ethanol, that treatment with the GNE-0877 inhibitor modulates the transcription of the target receptor genes and reduces the preference for ethanol in the animals of the Inflexible group. This result corroborates the hypothesis that the LRRK2 pathway is involved in the inflexible preference for ethanol behavior. Lastly, we identified a possible pharmacological target for the treatment of abusive preference behavior for ethanol.

oo

f

Introduction Ethanol addiction is a multifactorial disorder that was related to more than 3 million

pr

deaths in 2016 (WHO, 2016) .It is characterized by neurobiological and behavioral changes that intensify the seeking behavior and trigger adaptations that lead to the loss of control over

e-

the consumption of the drug (Pildervasser et al., 2014; Mathur et al., 2011a; Kily et al., 2008). The ethanol is a psychotropic drug with a broad and nonspecific activity that acts by

glutamatergic

different

brain

(Abrahao,

systems

such

as

Pr

modulating

Salinas and

dopaminergic,

opioid,

gabaergic,

and

Lovinguer, 2017). In the dopaminergic system,

al

dopamine is known to regulate motivational behaviors through the mesolimbic system. The modulation of this system by drugs of abuse such as the alcohol is associated with several

rn

public health problems (Baik, 2013), as it influences decision making and contributes to the

Jo u

compulsive use of the drug (Koob and Volkow, 2010). The highly conserved nature of the reward system, the universal capacity for drug abuse, and the known homology of these brain systems among species allows for the understanding of seeking behaviors in organisms other than mammals (Kily et al., 2008; Kedikian et al., 2013), which may facilitate the establishment of novel strategies for the treatment and prevention of alcohol use disorders. The zebrafish (Danio rerio) has been used as a model organism in numerous toxicology and drug addiction studies that sought to investigate the effects of ethanol on behavior (Gerlai et al., 2006; Ninkovic and Bally-Cuif, 2006; Mathur et al., 2011a; Tran et al., 2016a; Tran et al., 2016b) and on the neuromodulation of alcoholism related genes (Kily et al., 2008; Pan et al., 2011; Chaterjee et al., 2014; Tran et al., 2017a). In the context of behavioral changes linked to the motivational and reinforcement effects of alcohol, the Conditioned Place Preference (CPP) paradigm emerges. This test determines the preference for a reinforcing substance through the ability of learning and 2

Journal Pre-proof associating an environmental stimulus to the presence of the drug (Pavlovian conditioning) (Kedikian et al., 2013; Collier, 2014), thus resulting in preference or aversion. In zebrafish, for example, the ability of drugs of abuse to induce CPP has been proved using different substances such as cocaine (Darland and Dowling, 2001), nicotine (Kekidian et al., 2013), amphetamine (Ninkovic and et al, 2006), and opioids (Bretaud et al., 2007). In this species, it is known that preference acquisition may occur after a single exposure to ethanol and varies in a dose-dependent manner (Mathur et al., 2011a; Collier et al., 2014). In addition, acute ethanol exposure may reduce zebrafish anxious-type behavior, as evidenced by reduction of shoaling, locomotor activity, and time spent in depth (Gerlai et al., 2000; Egan et al, 2009;

oo

f

Mathur et al., 2011a; Tran et al., 2017b).

Besides the behavioral changes, the differential regulation in the transcript levels of

pr

ethanol molecular targets, such as the dopaminergic receptors type I and II (drd1 e drd2), is known to be involved in the transmission of the reinforcement effects and in the reinstatement

e-

of seeking behavior (Kily et al., 2008; Mathur et al., 2011a), and may be associated with motivated-related behaviors such as loss of control over ethanol consumption. In addition, the

Pr

glutamatergic and gabaergic pathways interact with the dopaminergic system, playing an important role in the behavioral responses following exposure to ethanol (Volkow, et al.,

al

2002a; De Witte, 2007; Tyacke et al., 2010). These pathways are often associated with the negative symptoms of withdrawal and with the establishment of dependence (Krystal et al.,

rn

2003; 2006). Therefore, the study of glutamatergic (grin1a e gria2a) and GABAergic

Jo u

(gabbr1a e gabbr1b) receptors is important for understanding the neuromodulation triggered by ethanol and for the elucidation of behaviors associated with type-addiction phenotypes. Our group has recently identified the involvement of the LRRK2 pathway in preference behavior and loss of control over ethanol consumption in mice (Silva e Silva et al., 2016). In this study, the major gene of this pathway, the Leucine-rich repeat kinase 2 (Lrrk2), was upregulated in mice that showed a preference for ethanol even after the presentation of an aversive stimulus (Inflexible phenotype). The lrrk2 gene encodes a complex and multifunctional protein that acts by modulating the Wingless (Wnt) and mitogen-activated protein kinase (MAPK) pathways, the calcium signaling cascade, and the MET signaling pathway, thus regulating processes such as synaptic maintenance, cell proliferation, and autophagy (Wallings et al., 2015). In the central nervous system (CNS), Lrrk2 is involved in dendritic spine formation, synaptic components reuptake, and synaptic plasticity (Shin et al., 2008; Beccano-Kelly et al., 2014; Cirnaru et al., 2014). In 3

Journal Pre-proof the context of Parkinson's disease, mutations that increase the kinase activity of this protein (G2019S and R1441C) are associated with the increased neuronal toxicity typical of familial cases of the disease (Ramsden et al., 2011; Sheng et al., 2012; Manzoni et al., 2013; Zhao et al., 2015). In this sense, the inhibition of Lrrk2 emerges as a protective measure against the neuronal toxicity induced by mutations in this gene. Pharmacological inhibitors of the kinase function of Lrrk2, which act mainly on the dephosphorylation of Ser910/935/1292 residues, have been developed (Wallings et al., 2015) with the aim of reestablishing the normal function of the protein (Estrada et al., 2013). Several inhibitors have been reported in the literature (West,

oo

f

2015; Atashrazm and Dzamko, 2016; Taymans and Greggio, 2016;). The selective inhibitor of LRRK2 kinase activity GNE-0877, previously tested on cells from transgenic mice

pr

expressing human LRRK2, acts to inhibit Ser1292 autophosphorylation. This highly potent inhibitor presents greater intramolecular flexibility, high selectivity, and capacity to penetrate

e-

the mammalian blood-brain barrier, which allows its use in pre-clinical and biosafety studies (Estrada et al., 2014) and makes GNE-0877 a candidate of interest for the treatment of alcohol

Pr

use disorders.

Herein, we used a population of zebrafish to develop a model applying CPP in the

al

identification of different ethanol preference phenotypes based on inter-individual differences. We characterized the model by evaluating the transcriptional regulation of six target genes

rn

involved in the motivational and rewarding effect of drug use: drd1, drd2, grin1a, gria2a,

Jo u

gabbr1a, and gabbr1b. In order to address the role of the LRRK2 pathway in the modulation of ethanol preference, we evaluated the behavioral response of the animals of the different phenotypic groups after treatment with the LRRK2 protein inhibitor GNE-0877. Our results revealed four phenotypes regarding the preference for ethanol - Light, Heavy, Negative Reinforcement, and Inflexible - and identified patterns of differential gene regulation in each of these groups. Treatment with the GNE-0877 inhibitor effectively modulated the preference behavior and indicated that the behavioral differences correlate with modifications in the regulation of dopaminergic, glutamatergic, and gabaergic receptors, as well as the lrrk2 gene. Materials e Methods 1. Animals and housing Eighty zebrafish with 20 days post fertilization (dpf) of both sexes, AB line (wild type), were obtained by crossing the stock acquired from the Zebrafish International Resource 4

Journal Pre-proof Center (ZIRC, USA) and kept at the animal facility of the Instituto Murciano de Investigación Biosanitária (Murcia, Spain). The mean weight of the animals used was 0.124g. During the acclimation phases, the animals were kept in acrylic aquariums with temperature and water quality control. For the CPP test, the animals were transferred to a rack specific for the species with water recirculation system and equipped with biological, chemical, and mechanical filters, as well as aeration and a ultraviolet light sterilization system. At all times, we kept a photoperiod of 14 hours of light and 10 hours of darkness, and the temperature was maintained between 26 and 27°C. The fish were fed twice daily with live food (artemia). All fish were kept following the welfare parameters for the species and the protocols were

oo

f

conducted according to the rules of the Ethics Committee on the Use of Animals (Comitê de Ética no Uso de Animais) of the Federal University of Minas Gerais, Minas Gerais, Brazil

e-

pr

(Protocol number 64/2016).

2. Experimental design

Pr

A schematic representation of the experimental design is presented in Figure 1. The study was divided into two experiments: I) Development and characterization of an ethanol

rn

using the GNE-0877 inhibitor.

al

preference model in zebrafish, and II) Functional study through a pharmacological approach

zebrafish.

2.1.1 Acclimation

Jo u

2.1 Experiment I. Development and characterization of an ethanol preference model in

During the acclimation period, the animals (n = 30) were kept in 10L (30 x 14 x 25 cm) tanks (density of 3 animals/liter) for eight days. Next, 24 hours before the CPP test, the animals were randomly individualized and numbered in 1.1L (12 x 8 x 12 cm) maintenance tanks and divided into a control group (Control, n = 10) and a group of acute exposure to ethanol (Acute, n = 20).

5

Journal Pre-proof 2.1.2 Conditioned Place Preference (CPP) To determine the ethanol preference phenotypes, we used an adaptation of the CPP test developed by Mathur et al. (2011b). The preference was evaluated in three moments: basal (B), post-conditioning (PC), and after withdrawal (AW). We used 3L (25 x 12 x 10 cm) experimental tanks with two patterns of texture in the bottom, i. e., half of the tank had a plain white bottom and the other half had a white bottom with evenly distributed black circles (environmental clue) (Mathur et al., 2011b). The experimental tanks were separated from each

f

other by isolators to prevent the animals from seeing the neighboring tank.

oo

 Determining the basal (B) preference

The previously individualized animals were carefully transported to the behavioral

pr

evaluation room (with controlled lighting, temperature and noise conditions.) and transferred to the experimental tanks being careful to minimize the stress due to the transference and

e-

hypoxia. Then, in the absence of ethanol, the animals were filmed for ten minutes to

Pr

determine the preference for each compartment. The basal (B) preference was defined as the side of the tank that the animal explored for longer (%).

al

 Conditioned exposure to the ethanol

rn

Following determination of the B preference, the animals were placed in the conditioning tanks, on the side opposite to the preference. These tanks were identical to those

Jo u

used to determine the preference, except for the presence of a sealed central divider to prevent the passage of water or drug, thus avoiding cross-contamination effects (Collier et al., 2014). On the least-preferred side, each animal from the Acute group was exposed to ethanol (Merck, Damstadt, Alemanha) at a concentration of 1% (v/v) for 20 minutes. Next, they were transferred to a tank containing water and kept for five minutes for the elimination of drug residues present in the body. Then, the fish were transferred to the side of B preference, which contained only water, and were held there for another 20 minutes. After the conditioning on both sides, the animals were transferred back and kept on the maintenance tanks until the following day, when the post-conditioning (PC) preference was determined. The same procedures were performed with the animals of the Control group without the addition of ethanol in the compartments.  Determining the post-conditioning (PC) preference 6

Journal Pre-proof The PC preference was determined following the same procedures described for establishing the B preference. Evaluation was performed 16 hours after the conditioning period to ensure the elimination of any residual effects of the drug administered on the previous day.  Determining the preference after withdrawal (AW) Following determination of the PC preference, the animals were kept in the maintenance tanks without ethanol for 16 days. At the end of this period, the post-withdrawal

oo

f

(AW) preference was established following the procedures previously described.

pr

2.1.3. Behavioral Assessment and Phenotyping

At the end of all phases of the CPP test, the animals were classified according to the

e-

individual preference for ethanol based on the filming records. The initial two minutes of filming were disregarded and the next five minutes were used for analysis in the behavioral

Pr

software EthoVision XT 14 (Noldus - Wageningen, Netherlands). This software generated data of average speed and total time spent by each animal on the white and dotted sides of the

al

tank at all times (B, PC, and AW). The preference results were expressed as the percentage of

rn

time spent on the side opposite to that of preference B (the side on which they were exposed to the ethanol). Percentages of time on the conditioning side that were higher than and

Jo u

statistically different from the hypothetical value of 50.1% were considered as a preference for ethanol. Values below and different from the threshold were considered as an aversion for the compartment. The mean velocity data were used to determine the locomotor activity of the animals. Animals with freezing behavior were excluded from all analyses.

2.1.4 Total RNA extraction By the end of the filming stages, animals were euthanized with an overdose of the anesthetic benzocaine (ethyl p-aminobenzoate, 250 mg/L) (Ross and Ross, 2009). The brains were dissected, immersed on a phosphate buffered saline solution (PBS), frozen in liquid nitrogen, and stored at -80°C. Total mRNA was extracted using Direct-zol RNA kits (Zymo Research, Irvine, EUA) according to the manufacturer's instructions. Samples were quantified using the DeNovix DS-11 (DeNovix, Delaware, EUA). All samples showed purity ratios of 7

Journal Pre-proof 260/280 and 230/260 between 1.8 and 2.2. RNA integrity was visualized on a 1.5% agarose gel stained with GelRed (Biotium, California, USA).

2.1.5 Primer design The exon sequences are available at the Ensembl Genome Browser database (www.ensembl.org/; accessed on October 6th , 2015). Primers were designed using the software Primer3 v.0.4.0 (Rozen and Skaletsky 2000). The quality and specificity of the oligonucleotides were tested using NetPrimer (www.premierbiosoft.com/netprimer/; accessed October

6th ,

2015)

and

Primer-BLAST

(www.ncbi.nlm.nih.gov/tools/primer-blast/;

f

on

oo

accessed on October 6th , 2015), respectively. All primers were positioned in inter-exon regions and synthesized by Sigma Aldrich, Spain. The sequences used are available in Table

e-

pr

1.

Pr

2.1.6 Reverse transcription and quantitative PCR

For each sample, 600ng of total mRNA were used for reverse transcription using IV VILO™ Master Mix (ThermoFisher Scientific), according to the

al

SuperScript™

manufacturer's instructions. Target gene transcripts were quantified by qPCR using the

rn

StepOne™ Real-Time PCR System (Applied Biosystems™) and SYBR Premix Ex Taq

Jo u

(Perfect Real Time, Takara). Amplification was conducted without the extension step (95°C for 10 minutes, followed by 40 cycles at 95°C for 15 seconds and 60°C for 1 minute). Fluorescence quantification was performed during the last step of the cycle (60°C). A negative control without a sample (NTC) was tested in all reactions. The qPCR data were analyzed by the Ct delta-delta method using the geometric mean of the reference genes eef1a1a (eukaryotic translation elongation factor 1 alpha 1a) and rpl13 (ribosomal protein L13) for normalization. Stability of the reference genes was confirmed using the algorithms BestKeeper and Genorm (Pfaffl et al. 2004, Wan et al. 2010). The relative amount of mRNA of the genes of interest was calculated as described by Vandesompele et al. (2002). 2.2 Experiment II. Functional study – a pharmacological approach using the inhibitor GNE0877. 8

Journal Pre-proof

For the functional study, the experimental model described above was repeated using 60 animals of similar age, lineage and with the same sex ratio, which were kept individualized and were randomly divided into Control (n = 10) and Acute (n = 40) groups.

2.2.1 Inhibitor administration To evaluate the effects of the Lrrk2 Ser1292 autophosphorylation inhibitor GNE-0877 (Cayman Chemical, Ann Arbor, Michigan, USA) on the zebrafish model, we performed a pre-test to determine the optimum experimental dosage and the time exposure that did not

oo

f

compromise the swimming behavior and the preference determination (Figure S1). A dose of 3nM was adopted, and the inhibitor was diluted according to the manufacturer's instructions.

pr

Following the pre-test, the phenotypic groups identified after determination of the AW preference were subdivided. For each phenotype, the individualized animals were subdivided

e-

into two groups: control (-) and inhibitor (+). Animals of the inhibitor group (+) were exposed to 3nM GNE-0877 dissolved in dimethylsulfoxide (DMSO). Animals of the control group (-)

Pr

were exposed to 1% DMSO only. Both DMSO and GNE-0877 were diluted in aqueous

al

solutions, and the animals were exposed by continuous submersion for three hours.

2.2.2 Preference after exposure to the inhibitor (AI)

rn

The preference after exposure to the inhibitor (AI) was determined following the same

Jo u

procedures described above for determination of the preference B. Evaluation was performed by filming the animals after three hours of exposure to the inhibitor GNE-0877. Following the filming,

animals

were

euthanized,

and

brain samples were collected

for

transcript

quantification of the same genes evaluated during the establishment of the model (Table 1).

3. Statistical analysis All data were analyzed for normality by the Shapiro-Wilk test. Using the RStudio statistical package, we performed: i) principal component analysis (PCA) to distinguish the phenotypes in relation to the ethanol preference; ii) Pearson correlation analysis among the moments of preference determination (B, PC, and AW); and iii) covariance analysis to evaluate the effect of the sex of the animals on determination of the preference behavior. The other analyses were conducted using the statistical package GraphPad Prism version 7.01. The 9

Journal Pre-proof validation analyses considering the number of animals that preferred the white or dotted side were performed by chi-square. To determine the existence of preference, i. e. if the mean time spent on the conditioning side was different from the hypothetical threshold of 50.1%, onesample t-test was performed. One-way ANOVA followed by a Tukey's post-hoc test was used to evaluate the mean time on each compartment side during the determination of B preference and the relative amount of mRNA for the target genes. In the evaluation of the locomotor activity at each moment (B, PC, and AW) and for each phenotype, we used a two-way repeated measures ANOVA followed by the Tukey’s test. In experiment II, one-sample t-test was used to evaluate the AI preference in relation

oo

f

to the 50.1% threshold. In addition, a t-test was used to evaluate: i) the behavioral differences within each phenotypic group, among animals that received (+) or did not receive (-) the

pr

treatment with the inhibitor; and ii) the effects of the inhibitor on the modulation of target transcripts among the animals of each phenotype. Two-way ANOVA was used to evaluate the

e-

modulation of the transcripts of each phenotype in relation to the animals in the Control group (with no exposure to ethanol) that were treated (+) or not (-) with the inhibitor.

Pr

In all analyses, data were expressed as the mean and standard error of the mean

rn

Results

al

(±SEM). Differences were considered significant when p ≤ 0.05.

Jo u

1. Development and characterization of a model of ethanol use in zebrafish 1.1 Phenotyping based on ethanol preference behavior In the CPP test, no significant B preference for any of the compartments (white or dotted) that could influence the outcome was observed (p > 0.05). Considering the total number of animals from both experiments (I and II), we observed 34 animals that initially preferred the white side and 42 that preferred the dotted side. The animals subjected to the CPP test and that were exposed to the ethanol (Acute group) were classified into four phenotypes according to their preference for the drug: 1) Light, animals that did not prefer the conditioning side at any time (B, PC, and AW); 2) Heavy, animals that preferred the side where the conditioning to the ethanol occurred in PC but lost preference in AW; 3) Negative Reinforcement, animals that did not alter their PC preference in relation to B, but began to seek the conditioning side in AW; and 4) Inflexible, animals that preferred the conditioning side in both PC and AW (Table 2 and Figure 2). 10

Journal Pre-proof In the Control group (with no exposure to the ethanol), no statistical differences were observed in relation to the threshold preference in PC (p = 0.4077) or AW (p = 0.3313), thus confirming the absence of intrinsic preference (Figure 2A). Animals of the Light phenotype presented a mean significantly lower than the preference threshold (50.1%) in B (p = 0.0001), PC (p = 0.0016), and AW (p = 0.0002) (Figure 2B). For animals of the Heavy phenotype, we observed a mean value statistically different and higher than 50.1% in PC (p < 0.0001) and statistically different and lower than the same threshold in AW (p = 0.0004) (Figure 2C). In the Negative Reinforcement group, the mean was statistically lower than and different from 50.1% in PC (p = 0.0075) and was higher than and different from the same threshold in AW

oo

f

(p = 0.0002) (Figure 2D). Finally, animals of the Inflexible phenotype showed a mean higher than and different from 50.1% in PC (p = 0.0052) and in AW (p = 0.0079) (Figure 2E). preference was presented in the

pr

Results that address the percentage of changed Supplementary figure 2 (S2).

e-

No significant differences in locomotor activity were observed among the moments of B, PC, and AW preference determination or among the phenotypes (p > 0.05). There was also

Pr

no effect of the sex of the animals on the preference behavior at any of the moments (p >

al

0.05).

rn

1.2 Analysis of clustering phenotypes

PCA analysis considering the variance of the preference data and the effects of the

Jo u

determination moments (B, PC, and AW) corroborated the grouping of the animals of experiments I and II into the four observed phenotypes (Figure 3). In this analysis, we used only the data of the animals that were exposed to ethanol (Acute group). We opted for this approach as we were aiming to evaluate and identify different phenotypes of preference to the ethanol in each of the moments of preference determination, excluding the effects related to the presence of the alcohol by itself. As ethanol was used as a tool to distinguish individuals with regards to their preference, the exclusion of the Control group in this clustering analysis is justified as these animals did not undergo treatment and, therefore, could belong to any of the phenotypes. We observed that each phenotype directly correlated to one of the preference determination moments (B, PC or AI), with no significant correlations being observed among the moments (Figure 3). Among the animals of the Heavy phenotype, PC had a greater 11

Journal Pre-proof influence on the distribution of the data, as this was the only moment in which the animals of this group preferred the side of exposure to the ethanol. The Negative Reinforcement phenotype data were justified by the AW variable, which was expected as this is the only moment of preference determination in which these animals preferred the exposure side. For the Inflexible phenotype, the variable that most explained the distribution of the data was B, since this was the moment when these animals altered their preference, which was later maintained in PC and AW. In addition, the Inflexible group was the phenotype that presented the smallest variation among the data when compared to the other phenotypic groups. The animals of the Light phenotype stood outside the area of correlation with the variables and

oo

f

were not influenced by the moments of evaluation since these animals showed no change in preference over the experimental protocol.

pr

An area of intersection, in which the data variances were approximated, was observed among all the phenotypes. This overlapping occurred close to the variable B and is justified

e-

as, at this moment, the preference behavior was the same among the phenotypic groups (i. e., there had been no exposure to ethanol that would allow the distinction of the four

Pr

phenotypes).

al

1.3 Brain transcriptional regulation of ethanol target receptors among preference phenotypes

rn

Figure 4 shows the transcript levels of ethanol target receptors in the zebrafish brain for each phenotype following acute exposure. In this first analysis of transcripts, we

Jo u

considered only animals from the experiment I. Among the phenotypic groups, we observed a significant difference in the transcripts levels of the dopamine type 1 (drd1) (F (4,21) = 11.87, p < 0.0001) and type 2 (drd2) (F (4, 21) = 15.64, p < 0.0001) receptors, the ionotropic NMethyl-D-aspartic acid (NMDA) (grin1a) (F (4,21) = 11.64, p = 0.0019) and α-amino-3hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) (gria2a) glutamate receptors (F (4, 22) = 50.69, p < 0.0001), and one of the GABA B subunits evaluated (gabbr1b) (F (4, 22) = 21.63, p < 0.0001). Post-hoc analyses revealed that drd1 is upregulated in animals of the Inflexible phenotype in comparison with all the others (Figure 4A). For drd2 and grin1a, we observed a downregulation in animals of all phenotypic groups in comparison to the Control (Figures 4B and C, respectively). On the other hand, the genes gria2a and gabbr1b were upregulated in all phenotypes in comparison to the Control (Figures 4D and E, respectively). In experiment I, no significant differences were observed in the transcript levels of the subunit 12

Journal Pre-proof a of the de gamma-aminobutyric acid type B receptor (gabbr1a) (F (4, 22) = 0.7788, p = 0.5509) (data not shown). One-way ANOVA pointed a group-specific effect on transcription of the central gene of the LRRK2 pathway: lrrk2 (F (4,21) = 5.599, p = 0.0031). Analysis of multiple comparisons by Tukey showed upregulation of this gene in the animals of the Inflexible group when compared with all other phenotypes after acute exposure (Figure 4F).

oo

2.1 Lrrk2 inhibitor as a modulator of preference behavior

f

2. Functional study using a pharmacological approach with GNE-0877 inhibitor

The effects of the exposure to GNE-0877 observed in the different preference

pr

phenotypes are represented in Figure 5. For evaluation of the effectiveness of the inhibitor in modulating the preference in each phenotype, two analyses were performed: i) comparison

e-

between animals that received (+) and did not receive (-) the treatment with the inhibitor; and ii) evaluation of the percentage of time spent on the conditioning side in AI in relation to the

Pr

hypothetical preference threshold (50.1%) in the animals of both groups (+) and (-). We did not observe differences between the groups (+) and (-), nor in relation to the

al

threshold (i. e., lack of preference) in the Control (no exposure to ethanol) and the Light phenotype groups (p > 0.05) (Figures 5A and B, respectively). In the Heavy phenotype, we

rn

observed a significant reduction (p = 0.0342) in the time spent by the group (+) on the side of

Jo u

exposure to ethanol in comparison to the group (-). However, when both groups (+ and -) were compared to the 50.1% threshold, the difference disappeared, which indicates the lack of preference or aversion (p > 0.05) (Figure 5C). For the Negative Reinforcement phenotype, we also observed no differences between the treated (+) and untreated (-) groups, and neither showed a difference to the threshold (p > 0.05) (Figure 5D). Lastly, in the Inflexible group, a reduction in the preference for the side of exposure to the ethanol was observed in the animals that were treated with the inhibitor (+) in comparison with those that were not (-) (p = 0.0148). Nevertheless, the reduction was insufficient to cause an aversion to the compartment in which the drug was received, as in both groups (+ and -), the time spent on the exposure side was higher than and different from 50.1% (p = 0.0360 and p = 0.0012, respectively) (Figure 5E).

13

Journal Pre-proof 2.2 Effects of Lrrk2 inhibition on the neuromodulation of the gene lrrk2 and on ethanol receptors targets Finally, we evaluated the effect of the selective inhibitor of Lrrk2 autophosphorylation (GNE-0877) on the transcriptional regulation of lrrk2 and other target genes generally related to the use of ethanol. Initially, we compared the animals within the same phenotypic group which received (+) or did not receive (-) treatment with the inhibitor (Figure 6). For the Light phenotype, we observed upregulation of drd2 (p = 0.0172) and downregulation of gria2a (p = 0.0193) in the animals (+) in comparison with the (-) (Figure 6A). In the Heavy phenotype,

f

we observed upregulation of drd2 (p = 0.0008) in the treated (+) group when compared to the

oo

untreated (-) (Figure 6B). In the Negative Reinforcement phenotype, on the other hand, upregulation of both drd2 (p = 0.0004) and grin1a (p = 0.0074) was observed in the group (+)

pr

compared with the (-) (Figure 6C). Animals with the Inflexible phenotype showed downregulation of drd1 (p < 0.0001), gabbrr1b (p = 0.0118), and lrrk2 (p = 0.0254) and

e-

upregulation of drd2 (p = 0.0222) in the treated (+) group in comparison with the untreated (-)

Pr

(Figure 6D).

Next, we compared the differences in transcriptional regulation for each phenotype to the Control group (without exposure to ethanol) that received (C+) or did not receive (C-)

al

treatment with the GNE-0877 inhibitor (Figure 7). In this analysis, significant differences

rn

were only observed in the Inflexible group. Differences were observed in the transcript levels of drd1 (F (1,19) = 65.86; p < 0.001) (Figure 7A) and lrrk2 (F (1,18) = 31.54; p < 0.0001)

Jo u

(Figure 7B), with both genes being upregulated in the animals of the Inflexible phenotype that did not receive treatment with the inhibitor (-) in comparison with the two Control groups (C+ and C-).When we compared the groups that received treatment (C+ and Inflexible+), only drd1 was downregulated in the group of Inflexible animals in comparison with C+ (p = 0.0231) (Figure 7A).

Discussion In the present study, we characterized four ethanol preference phenotypes (Light, Heavy, Negative Reinforcement, and Inflexible) in a heterogeneous zebrafish population using the CPP test. Although other protocols, such as free choice, have already been used in the characterization of different preference phenotypes in mice and rats (Silva e Silva et al., 2016; Pildervasser et al., 2014; Ribeiro et al., 2012; Short et al., 2006; Wolffgramm and 14

Journal Pre-proof Heyne, 1995), all the previous studies have used protocols of chronic exposure to ethanol. This is the first work in which phenotypes of preference are described in zebrafish and using an acute model. The phenotypes with no preference (Light) and with preference at some point during the experiment (Heavy and Inflexible) were named based on similar phenotypes previously described in mice (Silva e Silva et al., 2016). Additionally, we identified a new phenotypic category, which we named Negative Reinforcement, composed of animals that only preferred the side of exposure to ethanol (conditioning side) after withdrawal, thus characterizing a possible reinforcement potential of the drug in these animals following withdrawn.

oo

f

With this model, it was possible to evaluate the effects of acute ethanol exposure on preference determination in zebrafish. Mathur et al. (2011a) stated that a single exposure to

pr

ethanol is sufficient to induce CPP in zebrafish. Indeed, phenotypes of preference for ethanol (Heavy, Negative Reinforcement, and Inflexible) were observed in the animals after a single

e-

exposure of 20 minutes, thus corroborating the reinforcement potential of this substance (Figure 2).

Pr

The observed behavioral changes correlated with transcriptional modulation of CNS genes commonly associated with addiction-like phenotypes (Kily et al., 2008; Pan et al.,

al

2011; Klee et al., 2013). Upregulation of drd1a was observed in the Inflexible group in comparison with the other phenotypes, and downregulation of drd2a was detected in all

rn

groups of phenotypes in comparison to the Control. These results may indicate a motivational

Jo u

effect, with an increase of excitatory signaling via drd1 in animals with a high preference for ethanol (Inflexible) and a decrease of inhibitory signaling in all the animals exposed to the substance. This is in agreement with studies that detected an increase in the activity of the enzyme tyrosine hydroxylase and in the levels of dopamine and 3,4-dihydroxyphenylacetic acid (DOPAC) in the brain of zebrafish following an acute exposure to ethanol, thus suggesting that the activation of this pathway of neurotransmission plays an important role in the ability of the substance to induce CPP in this species (Chatterjee and Gerlai., 2009; Puttonen et al., 2013; Collier et al., 2014; Chatterjee et al., 2014; Tran et al., 2017a). Moreover, phenotypes of ethanol dependence have been associated with reduced levels of drd2 in humans (Volkow et al.,2002b), and overexpression of these receptors in mice is linked to the ability to decrease self-administration (Thanos et al., 2001). Studies that have used positron emission tomography (PET) to clarify the brain circuits involved with phenotypes of alcohol use have confirmed the relationship between the ability of drugs such 15

Journal Pre-proof as the ethanol to modulate dopaminergic pathways and their reinforcing effects on the human brain (Volkow et al., 2010; Hou et al., 2012). We additionally observed downregulation of grin1a and upregulation of gria2a and gabbr1b in all the phenotypes when compared with the Control group. The downregulation of the receptors grin1a in animals that have been exposed to the ethanol directly relates to the antagonistic effect that ethanol exerts on these receptors, reducing the glutamatergic excitatory neurotransmission via NMDA (Krystal et al.,2003). The upregulation of gria2a is consistent with other studies that have associated deregulation of this glutamatergic pathway with the motivational effect of the ethanol and with dependence phenotypes (Mead e

oo

f

Stephens, 2003; Kily et al., 2008). Lastly, the increased transcription of gabbr1b receptors observed in the all phenotypes exposed to ethanol is usually associated with the reduction of

pr

the consumption and preference for this substance (Vilas Boas et al., 2012; Tanchuck et al., 2012; Chester et al., 1999). This association may explain the behavior of the animals with the

e-

Light and Heavy phenotypes, which had no preference for ethanol or quit preferring the substance after the withdrawal. This, however, is not observed in the Negative Reinforcement

Pr

and Inflexible groups, which suggests that other signaling pathways may interact in the modulation of addiction- like behaviors.

al

In a previous study conducted by our group, Silva e Silva et al. (2016) observed differential transcriptional regulation of the Lrrk2 gene in mice with high preference and

rn

compulsive-like behavior associated with chronic ethanol consumption. Although herein we

Jo u

adopted a model of acute exposure, contrasting to the previous model, we also observed differential transcriptional regulation of this gene. lrrk2 was upregulated in animals with Inflexible phenotype in comparison with all the other phenotypes, following acute exposure to ethanol. This may indicate that the transcription of this gene is a key aspect for the acquisition and development of the Inflexible preference behavior, since, following a single exposure to ethanol, we observed an upregulation of this gene in the Inflexible group, which was maintained after 16 days in the absence of the substance (as observed during the re-exposure to the environmental clue previously associated with the drug). The LRRK2 pathway is involved in neuromodulation through the formation of dendritic spines, reuptake of synaptic components, and synaptic plasticity (Beccano-Kelly et al., 2014; Cirnaru et al., 2014; Shin et al., 2008). In this context, a correlation between lrrk2 and drd1 has been described, in which the Lrrk2 protein, an A-kinase anchoring protein-like (AKAP-like), has the ability to regulate the subcellular distribution of protein kinase A (PKA) 16

Journal Pre-proof and the phosphorylation of its targets in response to the activation of drd1 receptors, thus influencing the glutamatergic neurotransmission (Parisadou et al., 2014). Additionally, Rassu et al. (2017) have reported the detrimental effect of Lrrk2 protein on the process of drd1 internalization and

the consequent alteration in signal transduction and dopaminergic

neurotransmission (Rassu et al, 2017). This relationship between lrrk2 and drd1 corroborates our findings, as we observed upregulation of the transcript levels of both genes only in the Inflexible group, which could explain the characteristic seeking behavior of these animals. Given the observed upregulation of lrrk2, its possible involvement in the zebrafish Inflexible preference behavior, and the known relationship between increased kinase activity

oo

f

of this protein and changes in the neuronal plasticity and brain toxicity (Ramsden et al., 2011; Sheng et al., 2012; Manzoni et al., 2013; Zhao et al., 2015), we used a selective inhibitor of

pr

Ser1292 autophosphorylation, a marker of LRRK2 kinase activity (Estrada et al., 2014), to better understand the involvement of this gene in the modulation of the Inflexible preference

e-

behavior. Although the effects of inhibition of Lrrk2 have been previously tested in zebrafish in the context of Parkinson’s Disease using different methodological approaches (Ren et al.,

Pr

2011; Sheng et al., 2010; Sheng et al., 2012; Estrada et al., 2014; Zhao et al., 2015; Prabhudesai et al., 2016), this is, to the best of our knowledge, the first time that Lrrk2

al

inhibition is used to study alcohol-related behaviors. In the present study, we observed a reduction in ethanol preference (as measured by

rn

the time spent on the compartment of conditioning exposure to ethanol) in animals of both the

Jo u

Heavy and Inflexible groups following treatment with the GNE-0877 inhibitor. Given the association between Lrrk2 kinase activity and its neurotoxic effect in the CNS (Ramsden et al., 2011; Sheng et al., 2012), the use of inhibitors such as GNE-0877 emerges as a protective alternative in an attempt to mitigate the deleterious effects caused by alterations in the activity of this protein. The reduced preference observed in the Heavy and Inflexible phenotypes after exposure to GNE-0877 could characterize a neuroprotective effect of this inhibitor against the abusive ethanol consumption and highlights the potential of Lrrk2 as a possible therapeutic target in the context of alcohol use disorders. Recently, another molecule, 5’-deoxyadenosylcobalamin (AdoCbl), a physiological form of vitamin B12, had its effectiveness in the allosteric control (inhibition) of Lrrk2 kinase activity proved in brain cells and tissue. Treatment with AdoCbl was able to prevent the neurotoxic effects caused by increased Lrrk2 activity in neuronal cells of rodents and other

17

Journal Pre-proof animal models, such as Caenorhabditis elegans and Drosophila melanogaster, presenting variants associated with Parkinson’s Disease (Schaffner et al., 2019). Regarding the transcriptional regulation following treatment with the inhibitor, we observed upregulation of drd2 and downregulation of gria2a in animals of the Light phenotype treated with the inhibitor (+) in comparison with the untreated (-) animals of the same group. In this group, such results could be responsible for maintaining the absence of preference after exposure to the inhibitor, since the regulation of both genes has been associated with a decrease in self-administration and reward-seeking behavior, and also in the reinforcement effect of the ethanol in others species (Thanos et al., 2001; Mead and Stephens,

oo

f

2003; Kily et al., 2008). In the Heavy group, a similar regulation of drd2 was observed among the animals treated with GNE-0877 (+) in comparison with those untreated (-), which

pr

supports the reduction of ethanol preference following exposure to the inhibitor. In the Negative Reinforcement group, in addition to the modulation of drd2, we observed

number and

e-

upregulation de grin1a in the animals that were exposed to the inhibitor (+). The increased activity of receptors of this type is commonly associated

with the

Pr

pathophysiological state of ethanol abstinence (Nagy, 2008), which reiterates a putative negative reinforcement potential in these animals when the drug is withdrawn.

al

In the Inflexible phenotype, a larger number of genes were modulated as a consequence of inhibitor exposure, we observed differential regulation of drd1, drd2,

rn

gabbr1b, and lrrk2. The neuromodulation observed in this phenotype strengthens the

Jo u

hypothesis that the genes drd1 and lrrk2 are involved in the inflexible preference behavior, as we observed a significant reduction on the transcription of both genes in the individuals treated with GNE-0877 and an associated reduction in the preference for ethanol, thus indicating a putative neuroprotective function of the inhibitor. The upregulation of drd2, on the other hand, may indicate an increase in the inhibitory activity of this receptor in the presence of GNE-0877. Altogether, these results may explain the reduced CPP observed (Figure 5E). Lobo et al. (2010) have shown that stimulation of drd1 receptors in brain areas related to motivation is directly associated with cocaine's ability to increase CPP in mice. On the other hand, inhibition of drd2 can increase the reinforcement effect of amphetamine (Durieuxet et al., 2009, 2011; Ena et al., 2011). Since drd1 and drd2 act in opposite ways in the context of addiction (Lobo and Nestler., 2011), the reduction of excitatory signaling via drd1 and the increased inhibitory signaling via drd2 seem to have culminated in reduced 18

Journal Pre-proof motivation

and

CPP

in our animals of the

Inflexible phenotype.

Lastly,

although

downregulation of GABA is commonly associated with an increase in the rewarding effects of drugs of abuse (Enoch et al., 2017; Petrakis et al., 2004), the downregulation of gabbr1b that we observed in the Inflexible phenotype could be associated with the vulnerability of these animals to addiction, as a CPP reduction was observed in this group. In conclusion, the model developed herein allowed the identification of different phenotypes of preference for ethanol in a population of zebrafish, based on the interindividual differences observed through a behavioral paradigm of CPP. With this model, we demonstrated a differential transcriptional regulation of the main neurotransmitter receptors

oo

f

that are targets of ethanol (drd1, drd2, grin1a, gria2a, and gabbr1b), which are likely associated with the behaviors observed in each phenotype. Moreover, we showed the

pr

involvement of the lrrk2 gene in the modulation of the behavior of inflexible preference for the ethanol. This was confirmed by a functional approach using a selective inhibitor of the

e-

kinase activity of this protein, which was able to attenuate the preference for this drug in animals of the Inflexible phenotype. Our results indicate a conservation of the mechanism of

Pr

action of LRRK2 between mouse and zebrafish. Additionally, the model developed here allows the investigation of the processes involved in alcohol use disorders, especially those

al

related to the reinforcement potential of this substance.

rn

DECLARATION OF INTEREST

Jo u

Declarations of interest: none.

This works was supported by grants from Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG APQ-02942-16). FAPEMIG had no role in the design, analysis or writing of this article. This manuscript was reviewed by a professional science editor and by a native English-speaking copy editor to improve readability.

References ABRAHAO, K. P.; SALINAS, A. G.; LOVINGER, D. M. Alcohol and the Brain: Neuronal Molecular Targets, Synapses, and Circuits. Neuron, v. 96, n. 6, p. 1223–1238, 2017. ATASHRAZM, F.; DZAMKO, N. LRRK2 inhibitors and their potential in the treatment of Parkinson’s disease: Current perspectives. Clinical Pharmacology: Advances and Applications , v. 8, p. 177–189, 2016.

19

Journal Pre-proof BAIK, J.-H. Dopamine Signaling in reward-related behaviors. Frontiers in Neural Circuits , v. 7, n. October, p. 1–16, 2013. BECCANO-KELLY, D. A. et al. LRRK2 overexpression alters glutamatergic presynaptic plasticity, striatal dopamine tone, postsynaptic signal transduction, motor activity and memory. Human Molecular Genetics, v. 24, n. 5, p. 1336–1349, 2015. BRETAUD, S. et al. A choice behavior for morphine reveals experience-dependent drug preference and underlying neural substrates in developing larval zebrafish. Neuroscience , v. 146, n. 3, p. 1109– 1116, 2007.

f

CIRNARU, M. D. et al. LRRK2 kinase activity regulates synaptic vesicle trafficking and neurotransmitter release through modulation of LRRK2 macro-molecular complex. Frontiers in molecular neuroscience , v. 7, n. May, p. 49, 2014.

oo

CHATTERJEE, D.; GERLAI, R. High precision liquid chromatography analysis of dopaminergic and serotoninergic responses to acute alcohol exposure in zebrafish. v. 200, n. 1, p. 208–213, 2009.

e-

pr

CHATTERJEE, D.; SHAM, S.; GERLAI, R. Chronic and acute alcohol administration induced neurochemical changes in the brain: comparison of distinct zebrafish populations. Amino Acids, v.46, n.4, p. 921-930, 2014.

Pr

CHESTER, J. A.; CUNNINGHAM, C. L. GABA(A) receptors modulate ethanol-induced conditioned place preference and taste aversion in mice. Psychopharmacology, v. 144, n. 4, p. 363–372, 1999. COLLIER, A. D. et al. Zebrafish and conditioned place preference: A translational model of drug reward. Progress in Neuro-Psychopharmacology and Biological Psychiatry, v. 55, p. 16–25, 2014.

rn

al

DARLAND, T.; DOWLING, J. E. Behavioral screening for cocaine sensitivity in mutagenized zebrafish. Proceedings of the National Academy of Sciences of the United States of America, v. 98, n. 20, p. 11691–6, 2001.

Jo u

DE WITTE, P. Imbalance between neuroexcitatory and neuroinhibitory amino acids causes craving for ethanol: From animal to human being studies. Translation of Addictions Science Into Practice , v. 29, p. 57–79, 2007. DENG, X. et al. Characterization of a selective inhibitor of the Parkinson’s disease kinase LRRK2. v. 7, n. 4, p. 203–205, 2011. DURIEUX, P. F. et al. D2R striatopallidal neurons inhibit both locomotor and drug reward processes. Nature Neuroscience , v. 12, n. 4, p. 393–395, 2009. DURIEUX, P. F.; SCHIFFMANN, S. N.; D’EXAERDE, A. DE K. Targeting Neuronal Populations of the Striatum. Frontiers in Neuroanatomy, v. 5, n. July, p. 1–9, 2011. EGAN, R.J. et al. Understanding behavioral and physiological phenotypes of stress and anxiety in zebrafish. Behav Brain Research, v. 205, p. 38–44, 2009. ENA, S.; DE KERCHOVE D’EXAERDE, A.; SCHIFFMANN, S. N. Unraveling the Differential Functions and Regulation of Striatal Neuron Sub-Populations in Motor Control, Reward, and Motivational Processes. Frontiers in Behavioral Neuroscience , v. 5, n. July, p. 1–10, 2011.

20

Journal Pre-proof ESTRADA, A. A. et al. Discovery of Highly Potent, Selective, and Brain-Penetrable Leucine- Rich Repeat Kinase 2 (LRRK2) Small Molecule Inhibitors. Journal of Medicial Chemistry, v. 2, p. 1–6, 2014. GERLAI, R.; LAHAV, M.; GUO, S.; ROSENTHAL, A. Drinks like a fish: zebra fish (Danio rerio) as a behavior genetic model to study alcohol effects. Pharmacology Biochemistry Behavior, v. 67, p. 773–782, 2000. GERLAI, R.; LEE, V.; BLASER, R. Effects of acute and chronic ethanol exposure on the behavior of adult zebrafish (Danio rerio). Pharmacology Biochemistry and Behavior, v. 85, n. 4, p. 752–761, 2006.

oo

f

HOU, H.; TIAN, M.; ZHANG, H. Positron emission tomography molecular imaging of dopaminergic system in drug addiction. Anatomical Record, v. 295, n. 5, p. 722–733, 2012. KEDIKIAN, X.; FAILLACE, M. P.; BERNABEU, R. Behavioral and Molecular Analysis of Nicotine-Conditioned Place Preference in Zebrafish. PLoS ONE, v. 8, n. 7, p. 1–12, 2013.

e-

pr

KILY, L. J. M. et al. Gene expression changes in a Zebrafish model of drug dependency suggest conservation of neuro-adaptation pathways. Journal of Experimental Biology, v. 211, n. 10, p. 1623 LP – 1634, 2 maio 2008.

Pr

KLEE, E. W. et al. Zebrafish: A model for the study of addiction genetics. Human Genetics, v. 131, n. 6, p. 977–1008, 2012.

al

KRYSTAL, J. H. et al. N-methyl-D-aspartate glutamate receptors and alcoholism: Reward, dependence, treatment, and vulnerability. Pharmacology and Therapeutics , v. 99, n. 1, p. 79–94, 2003.

rn

KRYSTAL, J. H. et al. γ-Aminobutyric Acid Type A Receptors and Alcoholism. Archives of General Psychiatry, v. 63, n. 9, p. 957, 2006.

Jo u

KOOB, G. F.; VOLKOW, N. D. Neurocircuitry of Addiction. Neuropsychopharmacology, v. 35, n. 1, p. 217–238, 2010. LOBO, D. S. S. et al. Association of functional variants in the dopamine D2-like receptors with risk for gambling behaviour in healthy Caucasian subjects. Biological Psychology, v. 85, n. 1, p. 33–37, 2010. LOBO, M. K.; NESTLER, E. J. The Striatal Balancing Act in Drug Addiction: Distinct Roles of Direct and Indirect Pathway Medium Spiny Neurons. Frontiers in Neuroanatomy, v. 5, n. July, p. 1– 11, 2011. MANZONI, C. et al. Inhibition of LRRK2 kinase activity stimulates macroautophagy. Biochimica et Biophysica Acta - Molecular Cell Research, v. 1833, n. 12, p. 2900–2910, 2013. MATHUR, P.; BERBEROGLU, M. A.; GUO, S. Preference for ethanol in Zebrafish following a single exposure. Behavioural Brain Research, v. 217, n. 1, p. 128–133, 2011a. MATHUR, P.; LAU, B.; GUO, S. Conditioned place preference behavior in Zebrafish. Nature protocols, v. 6, n. 3, p. 338–345, 2011b. 21

Journal Pre-proof MEAD, A. N.; STEPHENS, D. N. Involvement of AMPA Receptor GluR2 Subunits in StimulusReward Learning: Evidence from Glutamate Receptor gria2 Knock-Out Mice. The Journal of Neuroscience , v. 23, n. 29, p. 9500–9507, 2018. NAGY, J. Alcohol Related Changes in Regulation of NMDA Receptor Functions. Current Neuropharmacology, v. 6, n. 1, p. 39–54, 2008. NINKOVIC, J. et al. Genetic Identification of AChE as a Positive Modulator of Addiction to the Psychostimulant D-Amphetamine in Zebrafish. Interscience , v. 66, p. 677–686, 2006. NINKOVIC, J.; BALLY-CUIF, L. The Zebrafish as a model system for assessing the reinforcing properties of drugs of abuse. Methods, v. 39, n. 3, p. 262–274, 2006b.

oo

f

PFAFFL, M. W. et al. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper - Excel-based tool using pair-wise correlations. Biotechnology Letters, v. 26, n. 6, p. 509–515, 2004.

pr

PAN, Y. et al. Chronic alcohol exposure induced gene expression changes in the Zebrafish brain. Behavioural Brain Research, v. 216, n. 1, p. 66–76, 2011.

e-

PARISIADOU L. et al., LRRK2 regulates synaptogenesis and dopamine receptor activation through modulation of PKA activity, Nat.Neurosci. 17 (3) 367–376, 2014

Pr

PETRAKIS, I. L. et al. Altered NMDA glutamate receptor antagonist response in individuals with a family vulnerability to alcoholism. American Journal of Psychiatry, v. 161, n. 10, p. 1776–1782, 2004.

rn

al

PILDERVASSER, J. V. N.; ABRAHAO, K. P.; SOUZA-FORMIGONI, M. L. O. Distinct behavioral phenotypes in ethanol-induced place preference are associated with different extinction and reinstatement but not behavioral sensitization responses. Frontiers in Behavioral Neuroscience, v. 8, n. August, p. 267, 2014.

Jo u

PRABHUDESAI, S. et al. LRRK2 knockdown in zebrafish causes developmental defects, neuronal loss, and synuclein aggregation. Journal of Neuroscience Research, v. 94, n. 8, p. 717–735, 2016. PUTTONEN, H. A J. et al. Acute ethanol treatment upregulates th1, th2, and hdc in larval Zebrafish in stable networks. Frontiers in neural circuits , v. 7, n. May, p. 102, 2013. RIBEIRO, A. F. et al. A transcriptional study in mice with different ethanol-drinking profiles: Possible involvement of the GABAB receptor. Pharmacology Biochemistry and Behavior, v. 102, n. 2, p. 224–232, 2012. RAMSDEN, N. et al. Chemoproteomics-based design of potent LRRK2-selective lead compounds that attenuate Parkinson’s disease-related toxicity in human neurons. v. 185, n. 2, p. 974–981, 2011. RASSU, M. et al. Role of LRRK2 in the regulation of dopamine receptor trafficking. PLoS ONE, v. 12, n. 6, p. 1–22, 2017. ROSS, L. G.; ROSS, B. Anaesthesia and Legislation. In: Anaesthetic and Sedative Techniques for Aquatic Animals, Third Edition, 2009. ROZEN, S.; SKALETSKY, H. Primer3 on the WWW for General Users and for Biologist Programmers. Methods in Molecular Biology, v. 132, n. 1, p. 365–386, 2000. 22

Journal Pre-proof

SCERBINA, T.; B.SC., D. C.; ROBERT, G. Dopamine receptor antagonism disrupts social preference in zebrafish, a strain comparison study. v. 43, n. 5, p. 2059–2072, 2012. SCHAFFNER, A. et al. Vitamin B 12 modulates Parkinson’s disease LRRK2 kinase activity through allosteric regulation and confers neuroprotection. Cell Research, v. 29, n. 4, p. 313–329, 2019. SHENG, D. et al. Deletion of the WD40 domain of LRRK2 in zebrafish causes parkinsonism-like loss of neurons and locomotive defect. PLoS Genetics, v. 6, n. 4, 2010. SHENG, Z. et al. Ser1292 autophosphorylation is an indicator of LRRK2 kinase activity and contributes to the cellular effects of PD mutations. Science translational medicine , v. 4, n. 164, 2012.

oo

f

SHIN, N. et al. LRRK2 regulates synaptic vesicle endocytosis. Experimental Cell Research, v. 314, n. 10, p. 2055–2065, 2008.

pr

SHORT, J. L.; DRAGO, J.; LAWRENCE, A. J. Comparison of ethanol preference and neurochemical measures of mesolimbic dopamine and adenosine systems across different strains of mice. Alcoholism: Clinical and Experimental Research, v. 30, n. 4, p. 606–620, 2006.

e-

SILVA E SILVA, D. A. et al. Inflexible ethanol intake: A putative link with the Lrrk2 pathway. Behavioural Brain Research, v. 313, p. 30–37, 2016.

Pr

TANCHUCK, M. A. et al. Assessment of GABA-B, Metabotropic Glutamate and Opioid Receptor Involvement In An Animal Model of Binge Drinking. v. 46, n. 2, p. 220–231, 2010.

al

TAYMANS, J.-M.; GREGGIO, E. LRRK2 Kinase Inhibition as a Therapeutic Strategy for Parkinson’s Disease, Where Do We Stand? Current Neuropharmacology, v. 14, n. 3, p. 214–225, 2016.

rn

THANOS, P. K. et al. Overexpression of dopamine D2 receptors reduces alcohol self-administration. Journal of Neurochemistry, v. 78, n. 5, p. 1094–1103, 2001.

Jo u

TRAN, S. et al. Interaction between handling induced stress and anxiolytic effects of ethanol in Zebrafish: A behavioral and neurochemical analysis. Behavioural Brain Research, v. 298, p. 278– 285, 2016a. TRAN, S.; FACCIOL, A.; GERLAI, R. Home tank water versus novel water differentially affect alcohol-induced locomotor activity and anxiety related behaviours in Zebrafish. Pharmacology Biochemistry and Behavior, v. 144, p. 13–19, 2016b. TRAN, S. et al. Acute alcohol exposure increases tyrosine hydroxylase protein expression and dopamine synthesis in Zebrafish. Behavioural Brain Research, v. 317, p. 237–241, 2017a. TRAN, S. et al. Zebrafish Are Able to Detect Ethanol in Their Environment. Zebrafish, v. 00, n. 00, p. 1-7, 2017b. TYACKE, R. J. et al. GABAB receptors in addiction and its treatment. Advances in Pharmacology, v. 58, n. C, p. 373–396, 2010. VANDESOMPELE, J. et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol, v. 3, n. 7, p. RESEARCH0034, Jun 18 2002. 23

Journal Pre-proof

VILLAS BOAS, G. R. et al. GABAB receptor agonist only reduces ethanol drinking in light-drinking mice. Pharmacology Biochemistry and Behavior, v. 102, n. 2, p. 233–240, 2012. VOLKOW, N. D. et al. Brain DA D2 receptors predict reinforcing effects of stimulants in humans: Replication study. Synapse, v. 46, n. 2, p. 79–82, 2002a. VOLKOW, N. D. et al. Effects of alcohol detoxification on dopamine D2 receptors in alcoholics: A preliminary study. Psychiatry Research - Neuroimaging, v. 116, n. 3, p. 163–172, 2002b. VOLKOW, N. D. et al. Imaging dopamine’s role in drug abuse and addiction. Neuropharmacology, v. 56, n. 2, p. 3–8, 2010.

oo

f

WALLINGS, R.; MANZONI, C.; BANDOPADHYAY, R. Cellular processes associated with LRRK2 function and dysfunction. FEBS Journal, v. 282, n. 15, p. 2806–2826, 2015.

pr

WAN, H. et al. Selection of appropriate reference genes for gene expression studies by quantitative real-time polymerase chain reaction in cucumber. Analytical Biochemistry, v. 399, n. 2, p. 257–261, 2010.

e-

WEST, A. B. Ten years and counting: Moving leucine-rich repeat kinase 2 inhibitors to the clinic. Movement Disorders, v. 30, n. 2, p. 180–189, 2015.

Pr

WOLFFGRAMM, J.; HEYNE, A. From controlled drug intake to loss of control: the irreversible development of drug addiction in the rat. Behavioural Brain Research, v. 70, n. 1, p. 77–94, 1995.

Jo u

rn

al

ZHAO, J. et al. LRRK2 dephosphorylation increases its ubiquitination. Biochemical Journal, v. 469, n. 1, p. 107–120, 2015.

24

Journal Pre-proof TABLES

drd1a drd2a gria2a grin1a gabbr1a gabbr1b

PRIMER REVERSE (5’-3’)

AMPLICON

ribosomal protein L13 eukaryotic translation elongation factor 1 alpha 1a

T CT GGAGGACT GT AAGAGGT

AGACGCACAAT CT TGAGAGCAG

148

CT ACT CTTCTTGATGCCCTTGAT

T GT CT CCAGCCACAT T ACCAC

309

CT CAAAT GT GGACT CGGAAAG AT ACTTCCGCT CTTTGGAT GAA

GAGAGAAGAGT T AGCCCAT CCA AT CAGGT AGTTGGT GGT GGT CT

226 275

T CACT GTGGAGAGAAT GGT GTC

GAT AAGCGT ATTTGCCT T T GGA

237

CACCAGGAT GT CCAT TTATTCA

CCT T AGGT CCTCTCTTGTTGTCA

269

GAGT GT CT GGT CATGTCGTGTT

T T ATCACT T T GGT CT GGT CT GC

186

dopamine receptor D1a dopamine receptor D2a glutamate receptor, ionotropic, AMPA 2a glutamate receptor, ionotropic, N-methyl Daspartate (NMDA) 1a gamma-aminobutyric acid (GABA) B receptor, 1a gamma-aminobutyric acid (GABA) B receptor, 1b leucine-rich repeat kinase 2

AAGCAT CACT GAAGGACCATCT

GGGAGACACT T T CTGACCT T T G

306

GAT GCT ACT GGAAGACCT GCTC

AAGACCCACCAAACT AGGAT GA

346

pr

lrrk2

PRIMER FO RWARD (5’-3’)

f

eef1a1l1

GENE DESCRIPTIO N

oo

GENE SYMBOL rpl13a

e-

Table 1. Sequence of the qPCR primers and respective amplicon sizes. Reference (rpl13a and eef1a1l1) and target (drd1a, drd2a, gria2a, grin1a, gabbr1a, gabbr1b, and

al

N (EXP I AND II)

Light

Animals that did not prefer the ethanol conditioning side in any of the observations.

n = 16

Heavy

Animals that preferred the ethanol conditioning side at PC and quit preferring at AW.

n = 16

Negative Reinforcement

Animals that began to prefer the ethanol conditioning side only at AW.

Inflexible

Animals that preferred the ethanol conditioning side at both PC and AW.

rn

DESCRIPTION

Jo u

PHENOTYPE

Pr

lrrk2) genes.

n = 18 n = 15

Table 2. Description and number of samples (N) of each phenotypic group distinguished by the CPP test in experiments I and II.

25

Journal Pre-proof LEGENDS OF THE FIGURES Figure 1. Experimental design. Experiment I – Establishment and characterization of the model. Acclimation: animals were randomly divided into two groups (Control, n = 10; and Acute, n = 20) and maintained in the experimental environment for eight days for adaptation. After this period, on the experimental day 1, we began the CPP test, which lasted 16 days. For this test, the animals of both groups were individualized. Preferences were determined by analyses of filming records (10 min each) from three moments: Basal (B), Post-Conditioning (PC), and After Withdrawal (AW). Preference in B was measured to

oo

f

establish the intrinsic preference of each animal for the different compartments of the tank (white and dotted) in the absence of the drug. Following determination of the preference B,

pr

each animal of the Acute group was exposed to ethanol conditioning (1% v/v) on the least preferred side for 20 min, followed by another period of 20 min on the preferred side

e-

containing only water. During this phase, the animals of the Control group were similarly conditioned on both sides of the tank, but in the absence of ethanol. Sixteen hours after the

Pr

conditioned exposure, the PC preference was determined and, after 16 days (on day 17), we determined the AW preference. Later on the same day, the animals were classified into Light

al

(L), Heavy (P), Negative Reinforcement (N), and Inflexible (I), based on data from the three preference determination time points (B, PC, and AW). The animals were, then, euthanized

rn

and their brains collected for analysis of the transcripts by qPCR. Experiment II - Functional

Jo u

study with the Lrrk2 Ser1292 autophosphorylation inhibitor GNE-0877 (n = 60). For the experiment II (n=60), all the steps of experiment I were repeated and, after phenotype classification, the animals of each group (L, H, N, I, and Control) were subdivided into two groups: one that was treated with the inhibitor (+) at the dose of 3nM; and another that received DMSO 1% only (-). On day 17 the animals were euthanized, and their brains were collected for the analysis of transcripts by qPCR. Figure 2. Ethanol preference determined by the CPP test for each phenotypic group – Experiments I e II. Data from experiments I and II are represented as the percentage of time spent on the ethanol conditioning side during the determination of the preferences B, PC, and AW for each phenotype distinguished after a single exposure of 20 minutes. Moments of preference determination: B = Basal, before conditioning; PC = Post-Conditioning, 16 hours after conditioning; AW = After Withdrawal, 16 days after withdrawal. The preferences were 26

Journal Pre-proof calculated in relation to a hypothetical preference threshold of 50.1% (red line), with mean values higher than and different from 50.1% indicating a preference for the ethanol conditioning side and mean values below this threshold indicating an aversion to the conditioning compartment. The graphs represent the preference changes that characterize each phenotype: (A) In the Control group (n=20), no statistical differences to the preference threshold were observed in PC (p = 0.4077) or AW (p = 0.3313), thus confirming the absence of intrinsic preference. (B) In the Light group (n=16), the means were significantly lower than the preference threshold in B (p = 0.0001), PC (p = 0.0016), and AW (p = 0.0002). (C) The Heavy group showed mean values lower than and statistically different from 50.1% in B and

oo

f

AW (p = 0.0014 and p = 0.0004, respectively) and mean in PC statistically different and higher than the same threshold (p < 0.0001). (D) In the Negative Reinforcement group

pr

(n=18), the means in B and PC were statistically inferior and different from 50.1% (p = 0.0002 and p = 0.0075, respectively), while the mean in AW was superior and different from

e-

the same threshold (p = 0.0002). (E) The Inflexible group (n=15) presented a mean lower than and different from 50.1% in B (p = 0.0290), and mean values higher than and different from

Pr

the threshold in PC (p = 0.0052) and AW (p = 0.0079). Differences between the means of the groups and the hypothetical value of 50.1% were considered statistically significant when p <

al

0.05. Data are expressed as the mean and standard error of the mean (±SEM).

rn

Figure 3. Scree plot showing the distribution of preference data and their relation to the

Jo u

moments of preference determination (B, PC, and AW) – Experiments I and II. Data from experiments I and II were plotted for B, PC, and AW. Ellipses indicate the grouping of the data into four phenotypes (Light n=16, Heavy – n=16, Negative Reinforcement – n=18, and Inflexible – n=15) and the arrows indicate the moments of preference determination (B, PC, and AW). The proximity of the ellipses (phenotypes) to the arrows indicates which moment of preference determination best explains the data variance of that phenotypic category. Ellipses' width and size indicate the variability of the data. The direction of the arrows indicates the correlation between the variables. The Control group was not included in the scree-plot. Since the different phenotypes were determined according to ethanol preference, the animals in this group could belong to any of the phenotypes but could not be classified, as they had not been exposed to the substance.

27

Journal Pre-proof Figure 4. Relative amount of mRNA of ethanol target receptors and of the gene lrrk2 in the zebrafish brain after acute exposure – Experiment I. Quantification carried out after the CPP test and the distinction of the ethanol preference phenotypes (Control (n=8), Light (n=6), Heavy (n=4), Negative Reinforcement (n=4), and Inflexible (n=5)) following acute exposure. (A) drd1: upregulated in the Inflexible phenotype in comparison to all the other groups, p < 0.05; (B) drd2: downregulated in all the phenotypes that were exposed to ethanol in comparison to the Control, p < 0.05; (C) grin1a: downregulated in all the phenotypes that were exposed to ethanol in comparison to the Control, p < 0.05; (D) gria2a: upregulated in all the phenotypes that were exposed to ethanol in comparison to the

oo

f

Control, p < 0.05; (E) gabbr1b: upregulated in all the phenotypes that were exposed to ethanol in comparison to the Control p < 0.05. (F) lrrk2: upregulated in the Inflexible

pr

phenotype in comparison to all the others, p < 0.05. Statistical analyses were performed by one-way ANOVA followed by the Tukey’s post-hoc test. Data are expressed as the mean

e-

and standard error of the mean (±SEM). Statistically significant differences are represented

Pr

by *, p < 0.05.

Figure 5. Ethanol preference determined by the CPP test and the effects of the

al

inhibitor GNE-0877 for each phenotypic group – Experiment II. Data are represented

rn

as the percentage of time spent on the ethanol conditioning side during the determination of the preferences B, PC, and AW for each phenotype distinguished after a single exposure

Jo u

to ethanol and exposure to the inhibitor GNE-0877 for three hours (AI). Moments of preference determination: B = Basal, before conditioning; PC = Post-Conditioning, 16 hours after conditioning; AW = After Withdrawal, 16 days after withdrawal; AI = After Inhibition, after three hours of treatment with the inhibitor. The preferences were calculated in relation to a hypothetical preference threshold of 50.1% (red line), with mean values higher than and different from 50.1% indicating a preference for the ethanol conditioning side and mean values below this threshold indicating an aversion to the conditioning compartment. In AI, comparisons were also carried out between the control group exposed to 1% DMSO (-) and the group exposed to 3nM of the inhibitor GNE-0877 (+). The graphs represent the preference changes that characterize each phenotype. Differences in B, P, and AW are as described in Figure 2. In AI: (A) In the Control group, no statistical differences were observed between the animals that were treated with the inhibitor (+) (n=6) and those that were not (-) (n=4) (p < 0.05). (B) Similarly, in the Light 28

Journal Pre-proof group, no differences were observed between the treated (+) (n=6) and untreated (-) (n=4) animals (p > 0.05). (C) In the Heavy group, we observed a reduction in the time spent on the side of exposure to ethanol in the animals that were treated with the inhibitor (+) (n=8) in comparison with those untreated (-) (n=4) (p = 0.0014), but no difference in relation to the threshold was detected (p > 0.05). (D) The Negative Reinforcement group did not show any difference between the treated (+) (n=9) and untreated (-) (n=4) animals (p > 0.05). (E) The Inflexible group showed a reduction on the preference of the animals treated with the inhibitor (+) (n=6) in comparison to those untreated (-) (n=4) (p = 0.0148), but no difference in relation to the preference threshold was detected (p > 0.05). Statistical

oo

f

analyses between the groups (+) and (–) at AI were carried out by t-test. Data are expressed as the mean and standard error of the mean (±SEM). Statistically significant differences are

pr

represented by *, p < 0.05.

e-

Figure 6. Relative amount of mRNA of ethanol target receptors and of the gene lrrk2 in the zebrafish brain of each phenotype in the presence and absence of the inhibitor

Pr

GNE-0877 – Experiment II. Quantification carried out after distinction of the ethanol preference phenotypes and exposure to the inhibitor. (A) Light phenotype, upregulation of

al

drd2 (p = 0.0172) and downregulation of gria2a (p = 0.0193) in the group (+) (n=6) in comparison with the (-) (n=4). (B) Heavy phenotype, upregulation of drd2 (p = 0.0008) in

rn

the treated group (+) (n=8) in comparison to the untreated (-) (n=4). (C) Negative

Jo u

Reinforcement phenotype, upregulation of the genes drd2 (p = 0.0004) and grin1a (p = 0.0074) in the animals (+) (n=9) when compared with the (-) (n=4). (D) Inflexible phenotype, downregulation of the genes drd1 (p < 0.0001), gabbr1b (p = 0.0118), and lrrk2 (p = 0.0254), and upregulation of drd2 (p = 0.0222) in the group (+) (n=6) in comparison to the (-) (n=4). Statistical analyses between the groups (+) and (-) for each gene and phenotype were carried out by t-test. Data are expressed as the mean and standard error of the mean (±SEM). Statistically significant differences are represented by *, p < 0.05.

Figure 7. Relative amount of drd1 and lrrk2 mRNA following exposure to the inhibitor GNE-0877 in the animals of the Inflexible phenotype in comparison with the Control that was not exposed to ethanol – Experiment II. In this analysis, we observed 29

Journal Pre-proof significant differences in the transcriptional regulation of drd1 (A) and lrrk2 (B) between the animals of the Control group (unexposed to ethanol) and the Inflexible group. (A) drd1: upregulated in the animals of the Inflexible phenotype that were not treated with the inhibitor (-) (n=4) in comparison to the untreated (C-) (n=4) (p < 0.0001) and treated (C+) (n=6) (p < 0.0001) Controls; and downregulated in the treated Inflexible phenotype (+) (n=6) in comparison with the treated Control (C+) (p = 0.0231). No significant difference was observed when the treated Inflexible phenotype (+) was compared to the untreated Control (C-). (B) lrrk2: upregulated in the animals of the Inflexible phenotype that were not exposed to treatment with the inhibitor (-) in comparison with the animals of the

oo

f

untreated (C-) (p < 0.0001) and treated (C+) (p = 0.0473) Control groups. No significant difference was observed in the Inflexible phenotype treated with the inhibitor (+) in

pr

comparison with both (C+ and C-) Controls (p > 0.05). Statistical analyses were performed by two-way ANOVA followed by the Tukey’s post-hoc test. Data are expressed as the

e-

mean and standard error of the mean (±SEM). Statistically significant differences are

Pr

represented by *, p < 0.05.

al

Figure S1. Pre-test for determination of dosage and time of exposure to the inhibitor GNE-0877 in experiment II. Two doses (1.5 nM (n=6) and 3 nM (n=6)) and two times of

rn

exposure to the treatment (3 hours and 24 hours) were tested. We evaluated the effect of the inhibitor on (A) locomotor activity, (B) immobility, and (C) impaired swimming. (A) A

Jo u

reduction in locomotor activity was observed for both dosages after three hours of exposure to the inhibitor in comparison to the control (n=5) (p < 0.05). After 24 hours, no alterations were observed in this behavior (p > 0.05). (B) and (C) We did not observe significant effects of the inhibitor dosage or exposure time on immobility or impaired swimming (p > 0.05). Statistical analyses were performed by two-way ANOVA followed by the Tukey’s post-hoc test. Data are expressed as the mean and standard error of the mean (±SEM). Statistically significant differences are represented by *, p < 0.05.

Figure S2. Changes in preference for the conditioned compartment of control and each phenotype. Changed preference was calculated for each preference determination moments (PC, AW and AI, represented by each column). In the control group it was not found any change in the preference for PC, AW and AI. For the Light phenotype it was 30

Journal Pre-proof observed an increase in preference in AI when compared AW (p=0.0051). For Heavy phenotype it was saw a decrease in the preference in AW (p=0.0033) and AI (p=0.0013) compared to PC. For Negative Reinforcement group it was found an increase in preference in AW (p<0.0001) and AI (p=0.0243) compared to PC. In the Inflexible group it was not found any change in the preference for PC, AW and AI. Change in preference = (% time in the ethanol paired compartment after conditioning) − (% time in the same

Jo u

rn

al

Pr

e-

pr

oo

f

compartment before conditioning). Mean±SEM are shown.

31

Journal Pre-proof ETHICAL STATEMENT All fish were kept following the welfare parameters for the species and the protocols were conducted according to the rules of the Ethics Committee on the Use of Animals (Comitê de Ética no Uso de Animais) of the Federal University of Minas Gerais, Minas Gerais, Brazil

Jo u

rn

al

Pr

e-

pr

oo

f

(Protocol number 64/2016).

32

Journal Pre-proof Highlights 

Four distinct ethanol preference phenotypes (Light, Heavy, Negative Reinforcement, and Inflexible) based on the behavioral Conditioned Place Preference (CPP) paradigm;



Different transcriptional regulation patterns of the drd1, drd2, grin1a, gria2a, and gabbr1b receptors for each fenotype;



The lrrk2 gene was hyperregulated only in the brains of the animals with the Inflexible phenotype;



The treatment with the GNE-0877 inhibitor modulates the transcription of the target receptor genes and reduces the preference for ethanol in the animals of the Inflexible group;

oo

f

This result corroborates the hypothesis that the LRRK2 pathway is involved in the inflexible

rn

al

Pr

e-

pr

preference for ethanol behavior;

Jo u



33

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9