Abnormal brain development of monoamine oxidase mutant zebrafish and impaired social interaction of heterozygous fish

ABSTRACT Monoamine oxidase (MAO) deficiency and imbalanced levels of brain monoamines have been associated with developmental delay, neuropsychiatric disorders and aggressive behavior. Animal models are valuable tools to gain mechanistic insight into outcomes associated with MAO deficiency. Here, we report a novel genetic model to study the effects of mao loss of function in zebrafish. Quantitative PCR, in situ hybridization and immunocytochemistry were used to study neurotransmitter systems and expression of relevant genes for brain development in zebrafish mao mutants. Larval and adult fish behavior was evaluated through different tests. Stronger serotonin immunoreactivity was detected in mao+/− and mao−/− larvae compared with their mao+/+ siblings. mao−/− larvae were hypoactive, and presented decreased reactions to visual and acoustic stimuli. They also had impaired histaminergic and dopaminergic systems, abnormal expression of developmental markers and died within 20 days post-fertilization. mao+/− fish were viable, grew until adulthood, and demonstrated anxiety-like behavior and impaired social interactions compared with adult mao+/+ siblings. Our results indicate that mao−/− and mao+/− mutants could be promising tools to study the roles of MAO in brain development and behavior. This article has an associated First Person interview with the first author of the paper.


INTRODUCTION
Monoamine oxidases catalyze the oxidative deamination of several endogenous and dietary amines, including dopamine (DA), norepinephrine (NE), serotonin (5-HT) and the first histamine metabolite, tele-N-methylhistamine. In mammals there are two isoforms (MAO A and MAO B) that can be distinguished on the basis of their substrate specificity and their sensitivity towards specific inhibitors. MAO A deaminates 5-HT and NE, whereas MAO B deaminates phenethylamine (PEA) (Glover et al., 1977). DA is primarily metabolized by MAO A in rodents and by MAO B in humans (Bortolato et al., 2008). Clorgyline and deprenyl inhibit MAO A and B, respectively (Sullivan et al., 1986).
Several studies link both MAO forms to different brain disorders (Jones and Raghanti, 2021). A role for increased MAO A activity/expression in the pathophysiology of different forms of depression, such as Major Depressive Disorder and postpartum depression, has been established (Sacher et al., 2010;Schulze et al., 2000). A complete and selective deficiency of enzymatic activity of MAO A was described in Brunner syndrome patients (Brunner et al., 1993). They presented antisocial and violent conduct as a maladaptive response to environmental triggers, mild cognitive impairments, as well as stereotyped hand movements and parasomnias. Some reports indicate a role for MAO A in the pathophysiology of autism spectrum disorder (ASD) (Bortolato et al., 2018).
Particularly, low-activity MAO A variants have been linked to higher severity of social impairments boys with ASD (Cohen et al., 2011). Postmortem brain tissue from subjects with ASD were analyzed and a significant impairment in the activity of MAO A in the cerebellum and frontal cortex was detected (Gu et al., 2017). MAO A loss-of-function in mice leads to high levels of brain 5-HT and NE and aggressiveness (Cases et al., 1995a). MAO A KO mice also recapitulate the main core deficits observed in ASD, including social impairment and communication impairments (Bortolato et al., 2013). Additionally, MAO A mutant mice display stereotypic behavior across several tasks, including marble burying, hole-board exploration and spontaneous alternations in a T maze (Bortolato et al., 2013).
The single nucleotide polymorphism (SNP) rs1799836 of MAO B was selected for association analysis in 537 schizophrenia patients and 536 healthy controls and it was identified as a risk factor in the development of schizophrenia (Wei et al., 2011). In Alzheimer's disease, MAO B has been found to be increased in astrocytes and pyramidal neurons. Additionally, it plays a key role in amyloid β peptide formation (Schedin-Weiss et al., 2017). A strong association between the MAO B gene and adult attention deficit hyperactivity disorder (ADHD) was also identified (Ribasés et al., 2009), and reports indicate that deprenyl reduces ADHD symptoms. This effect of MAO B inhibition on ADHD symptoms may lie in the increased level of PEA, which is assumed to act as an endogenous amphetamine (Janssen et al., 1999). Interestingly, MAO B mutant mice display increased brain levels of PEA, but unaltered locomotor activity and do not display anxious-like behavior and, differently from MAO A mutant mice, do not show an aggressive behavior (Grimsby et al., 1997).
Zebrafish has become a popular model organism in neuroscience because its genes share homology with those of humans and it possesses all main neurotransmitters (Gerlai, 2011;Kalueff et al., 2014;Panula et al., 2010). Unlike mammals, zebrafish possess only one form of mao, which displays a strong affinity for 5-HT and a modest one for DA, and is inhibited by both clorgyline and deprenyl (Anichtchik et al., 2006). To our knowledge, a mao zebrafish mutant has not been described and characterized yet. The study of outcomes related to disruption of mao functioning in zebrafish development has been limited to the use of pharmacological inhibitors (He et al., 2013;Sallinen et al., 2009b;Setini et al., 2005). Although this approach is widely used and can provide valuable information, interpreting the results of zebrafish exposure to chemicals, more specifically at early development, can be challenging for several reasons. For example, there are wide variations in the duration of exposure, whether the animals are exposed individually or in groups, whether the chemical is renewed daily or not, the "window" of exposure and how soon after exposure the animal is assessed. Moreover, deprenyl can be metabolized to amphetamine, which could alter DA neurotransmission and generate effects independent of mao inhibition (Karoum et al., 1982).
We analyzed the monoaminergic systems, developmental markers and behavioral parameters of larval mao -/and mao +/zebrafish in comparison with their mao +/+ siblings. Furthermore, in adult and juvenile mao +/zebrafish, we analyzed parameters relevant for ASD, such as social behavior, anxiety, mao activity and gene expression.
In the acoustic/vibrational startle test (at 6 dpf), we observed that the response after the first stimulus did not differ between mao +/+ , mao +/and mao -/larvae (n=15 mao +/+ , n=16 for mao +/and n= 12 for mao -/-; two-way ANOVA, genotype effect F(2, 400) = 26.96, p < 0.01. Tukey's post hoc test significances indicated in the graph. Fig. 1F). Interestingly, mao +/larvae showed a significantly increased startle response compared with their siblings on the second, third, and fourth stimuli. Mao -/larvae habituated faster to repeated exposure of the acoustic/vibrational stimuli at 20 s inter-stimulus-interval (ISI), while their siblings did less so.

Mao +/adult fish display abnormal social interaction and weaker mao activity
Juvenile mao +/+ and mao +/fish (30 dpf) were evaluated in a social contact assay where a pair of fish of the same genotype were placed in an arena for 6 minutes (Fig. 5A). The total duration in proximity (at a distance ≤ 0.8 cm) was not different when mao +/+ and mao +/fish were compared (n=8 pairs for each genotype; p > 0.05, Fig. 5B). However, mao +/fish were less frequently in proximity to each other (n=8 pairs for each genotype; p < 0.05, Fig. 5C). Additionally, we evaluated the frequency of direct contact between the fishes in the arena, but no statistically significant difference was detected when genotypes were compared (n=8 pairs for each genotype; p > 0.05, Fig. 5D).
To test whether mao deficiency affects fish shoaling behavior, four 40 dpf fish per trial were placed in a cylindrical plastic container (Fig. 5E). The average distance between the test fish and the other three shoal members and total duration in proximity (the nearest interindividual distance defined as 1 cm) were analyzed. The time spent in proximity with shoal members was significantly shorter in the mao +/fish group than mao +/+ siblings (n = 4 trials for each genotype; p < 0.05, Fig. 5F). Furthermore, the interindividual distance was significantly greater in the mao +/fish group than mao +/+ sibling group (n = 4 trials for each genotype; p < 0.05, Fig. 5G). After the trial, the brains of fish from both genotypes were dissected, and the mao activity histochemical assay was performed (n=4 for each genotype; Fig. 5H and I). Mao +/brains showed a weaker mao activity when compared with the brains of mao +/+ siblings in the hypothalamus area.
Finally, the social preference of adult fish was tested. Figure 6A shows the traces of the swimming pattern of mao +/+ and mao +/adult fish during the social interaction test. Compared with their mao +/+ siblings, mao +/adult fish did not differ statistically in the time spent in the zone closest to the compartment with the stimulus fish (n=8 for each genotype; p > 0.05, Fig. 6B) and spent less time in the non-social zone (n=8 for each genotype; p < 0.05, Fig. 6C). However, mao +/adult fish preferred to stay longer in the distal area of the apparatus (n=8 for each genotype; p < Disease Models & Mechanisms • DMM • Accepted manuscript 0.05, Fig. 6D). When we compared the time spent in the social zone by the mao +/+ fish with the time spent in both distal zone and non-social zone summed, these animals showed a preference for the area closer to the social stimulus detected (n=8 for each genotype; p < 0.05, Fig. 6E), whereas when the same comparison was made with their mao +/siblings, no significant difference was detected.
Mao +/+ and mao +/adult fish display similar locomotor activity and thigmotaxis Schemes of the locomotor activity test with two digitized zones and representative movement traces are shown in Fig. 7A. Locomotor activity, measured as total distance moved, did not significantly differ between mao +/+ and mao +/adult fish during a 10 min trial (n=7 for each genotype; p > 0.05, Fig. 7B). No differences in velocity were detected (n=7 for each genotype; p > 0.05, Fig. 7C). During the same trial, we also assessed thigmotaxis, a behavior in which animals spend most of the time of a session near the walls of the apparatus (Maximino et al., 2010). No difference between the genotypes was detected in this behavior, with both mao +/+ and mao +/adult fish spending most of the trial in the zone close to the arena wall (n=7 for each genotype; p < 0.01, Fig. 7D).

Mao +/adult fish display anxiety-like behavior in the novel tank diving test
The initial preference of the zebrafish for the bottom of a novel tank, and subsequent exploration of the rest of the tank, is commonly interpreted as precautionary antipredator response followed by alleviation of anxiety, respectively. We used a novel tank diving assay to study anxiety-related risk-taking behavior of mao +/+ and mao +/adult fish. The novel tank diving area was digitally divided into three zones, and representative swimming tracks are shown in Fig

Gene expression in mao +/adult zebrafish brains
We investigated the brains of mao +/adult fish to detect possible alterations that could be associated with their impaired behavior. SH3 and multiple ankyrin repeat domains 3 (shank3b) and methyl CpG binding protein 2 (mecp2) transcripts were quantified because of the extensive literature associating these genes with ASD (Muhle et al., 2004;Pardo and Eberhart, 2007), but no differences were detected when the genotypes were compared (n=4 for each genotype; p > 0.05, Fig. 8A and B). Histamine receptor h3 (hrh3) plays roles in anxiety and cognition, and recent

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findings suggest that this receptor could be a potential therapeutic target for ASD (Baronio et al., 2015;Eissa et al., 2018), but mao +/+ and mao +/presented similar levels of this transcript (n=4 for each genotype; p > 0.05, Fig. 8C). As expected, the mao +/brains displayed a significantly reduced level of mao mRNA compared with the brains of mao +/+ siblings (n=4 for each genotype; p < 0.01, Fig. 8D). We quantified vmat2 transcript levels to assess this key factor in monoaminergic neurotransmission (Fon et al., 1997). Vmat2 mRNA was upregulated in mao +/fish brains (n=4 for each genotype; p < 0.05, Fig. 8E). In zebrafish, serotonin transporter a (serta)-positive neurons are found in the raphe nuclei and the ventral posterior tuberculum. A similar distribution pattern of serta was detected in both mao +/+ and mao +/zebrafish (n=4 for each genotype; p > 0.05, Fig. 8G-J).
qPCR analysis using whole brains as samples indicated equal expression levels of serta mRNA in mao +/+ and mao +/zebrafish (n=4 for each genotype; p > 0.05, Fig. 8F).

DISCUSSION
Mao loss-of-function in mao -/zebrafish larvae led to hypoactivity and abnormal serotonergic, dopaminergic and histaminergic systems. The expression of important developmental markers was altered and mao -/larvae died within 20 dpf. Mao +/animals were viable, grew until adulthood and demonstrated anxiety-like behavior and impaired social interaction.
The hypoactive phenotype was detected during 24h basic locomotor activity evaluation where the distance moved by mao -/larvae was significantly decreased when compared with mao +/+ and mao +/siblings. This is in agreement with a previous report where mao inhibition in larval WT zebrafish decreases locomotion (Sallinen et al., 2009b). Additionally, weaker reactivity to acoustic/vibrational and visual stimuli was displayed by mao -/larvae. This could be a consequence of the severe hypoactive phenotype and general arrested development displayed by the mutants, considering that these responses are mediated by independent processes. The Mauthner cells modulate the escape response in zebrafish after acoustic/vibrational stimuli. After such stimuli, larvae will display a short latency response with an increase in locomotor activity and velocity. This phenotype is thought to be a form predation avoidance. A hyperactive phenotype is also noted when there is abrupt change in illumination, such as in the dark-flash response test. Initially, it was hypothesized that this was also an escape response to predation. However, it has been shown that this behavior is not mediated by the Mauthner cells and is more likely to be navigational rather than defensive (Burgess and Granato, 2007). Interestingly, mao +/larvae showed a difficulty to adapt to repeated acoustic/vibrational stimuli in comparison to mao +/+ and mao -/larvae. The startle response is associated with cognitive processing of sensory information, which is important for modeling cognitive deficits, resembling those associated with anxiety (Pittman and Lott, 2014). A similar

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result was obtained when dark-flash response was evaluated and mao +/larvae displayed a significantly stronger reaction than mao +/+ and mao -/larvae in the first dark flash. Additionally, compared with mao +/+ larvae, mao +/larvae took longer to adapt to the change in illumination and to return to the baseline activity.
Mao inhibition in larval WT zebrafish leads to increased levels of 5-HT (Sallinen et al., 2009b). When we quantified 5-HT-positive neurons in different serotonergic groups of the brain in mao -/larvae we verified a reduced number of 5-HT immunoreactive cells. However, a strong extracellular 5-HT immunoreactivity could be noted, as well as increased density of immunoreactive fibers. It is possible that the excess of 5-HT led to a toxic effect causing cell death.
Similar phenotype was reported when the brains of larval WT zebrafish treated with a mao inhibitor presented reduced 5-HT immunoreactivity in the serotonergic cell somata, extracellular 5-HT immunoreactivity and increased density of 5-HT-immunoreactive fibers (Sallinen et al., 2009b).
In situ hybridization showed a weaker tph1a signal in larval mao -/brains than in mao +/+ and mao +/brains. However, a significantly higher expression of this transcript was detected by RT-qPCR in whole mao -/whole larvae when compared with mao +/+ and mao +/siblings. One explanation for the discrepancy between these results is that for the RT-qPCR analysis pools of whole larvae were used as samples and tph1a is expressed in in skin and pharyngeal arch neuroepithelial cells (NECs) and nerves innervating NECs (Pan et al., 2021).
The likely neurotoxicity caused by the excess of 5-HT might have affected other monoaminergic populations and contributed to the early death of the mutant, as we detected reduced numbers of histaminergic and Th1-immmunoreactive neurons in the brains of mao -/larvae, when compared with their mao +/+ and mao +/siblings. Furthermore, the microglial marker apoeb was upregulated in mao -/larvae, raising the possibility of increased apoptosis in these larvae (Elliott et al., 2007).
It has been reported that zebrafish exposed to Trans-2- Signs of mild impairment in social behavior in juvenile mao +/fish were detected at 30 dpf when in a paradigm that evaluates social contact they were less frequently in proximity with another fish of same genotype. When shoaling behavior was evaluated at 40 dpf, we verified that juvenile mao +/fish in groups of 4 individuals spent less time in proximity and that there was an increased average distance between subjects when compared with the trials containing mao +/+ siblings. Shoaling behavior is an innate form of social interaction displayed by zebrafish that serves different purposes in their natural habitat (Pitcher, 1986). Additionally, mao +/adult zebrafish did not show a preference for a social stimulus when evaluated in a social interaction apparatus. These behavioral alterations could be a consequence of yet unknown developmental defects caused by mao deficiency in mao +/larvae, rather than a direct effect of mao deficiency in juvenile and adult fish.
Overall, mao +/zebrafish presented only a mild impairment in social behavior. This is a caveat that must be considered when using this research tool to model brain disorders where However, in a PET study of SERT binding in adults with Asperger's disorder no alterations were found (Girgis et al., 2011). Here we report no difference in the expression of serta in the brains of mao +/fish in comparison with mao +/+ siblings after whole brain qPCR analysis.
In summary, the present work contributes to previous pharmacological data concerning the behavioral and neurochemical consequences of mao inactivation in zebrafish. The monoaminergic systems regulate neuronal growth, differentiation, migration and survival. Thus, disrupted monoaminergic systems can lead to impairments in brain function and mental illness making mao -/fish a promising tool to study the roles of MAO and monoamines during brain development. Mao +/fish present mild impairment in social behavior and anxiety, and is a potential tool for studies that aim to assess the developmental and behavioral outcomes of interaction between environmental factors and MAO genotype.

Mao mutant
The mao mutant (sa 31732 ) was generated by the Sanger Institute Zebrafish Mutation Project and contains an A/T nonsense mutation at nucleotide 685 of the open reading frame, which is predicted to generate a 229-amino acid protein compared with the 522-amino acid WT protein.
Larvae were raised on 14:10 (light/dark, lights on at 8:00 A.M.) cycles at 28°C and fed daily once with flake food and two times with live artemia. Adult fish were raised in continuously cycling Aquatic Habitats Systems with complete exchange of water in each tank every 6-10 min.
Circulating water was UV-sterilized, and filtered with foam filters and activated charcoal. Water quality, including temperature (28 ± 0.5°C), pH value (7.4 ± 0.2), and conductivity (450 ± 10 mS), was monitored continuously. Embryos were obtained by natural spawning, collected from the breeding tanks, and staged in hours post-fertilization (hpf), days post-fertilization (dpf), or months post-fertilization (mpf) as previously described (Kimmel et al., 1995). The permits for all experiments were obtained from the Office of the Regional Government of Southern Finland, in agreement with the ethical guidelines of the European convention.
1 μL of Proteinase K (20 mg/mL) was added to remove protein, and the mixture was incubated at 55°C for at least 4 hours. To inactivate Proteinase K, samples were incubated at 98°C for 10 minutes and quenched on ice.
To detect mutations, high-resolution melting (HRM) curve acquisition and analysis was performed.
Primers flanking the mutation site were designed using Primer-BLAST and were as follow: Forward-AATGACAGGAGCGCAAGTTT and Reverse-GTAAACCTCCTCATTCACCGTC. Those with analogous melting curves were characterized as the same genotype. After genotyping, larval (6-10 dpf), juvenile (30-40 dpf) and adult (12 mpf) fish were raised and used in the experiments. All the comparisons were made between mutants and their wild-type siblings.

Behavioral assays
The samples used in a particular behavioral test were not included in other tests at different developmental stages. All adult individuals in a behavioral evaluation were male siblings and tests were repeated at least once with an independent biological replicate.

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24h locomotion tracking: At 6 dpf, mao larvae of each genotype were tracked simultaneously for 24 h, with the light conditions following the regular light/dark cycle of the larvae (Puttonen et al., 2018). The trial was started at 12:00 noon. The day and night activity was analyzed in 60 min bins by calculating the total distance moved. Larvae were individually tracked using the DanioVision (Noldus Information Technology, Wageningen, The Netherlands) system and EthoVision XT software in 48-well-plates. The diameter of the wells was 12 mm.
Larval dark-flash response: The dark-flash response of larvae was evaluated at 6 dpf as described before (Baronio et al., 2018). Briefly, after initial 5 min of basic locomotor activity tracking, larvae were exposed to alternating 2 min periods of darkness and light, and with three periods of darkness in total. The locomotor activity was analyzed in 30 s and 1s bins. Larvae were individually tracked in 48-well plates using the DanioVision system and EthoVision XT software (Noldus Information Technology, Wageningen, The Netherlands). This behavioral test was done between 12:00 and 16:00.
Larval acoustic/vibrational startle: This behavioral protocol has been described previously (Van Den Bos et al., 2017). Briefly, larvae (6 dpf) were transferred from Petri dishes to 48-well plates filled with 1 ml E3 medium. The protocol (lights on) consisted of 10 min acclimation, followed by 10 acoustic/vibrational stimuli (DanioVision intensity setting 6) with a 20 s inter-stimulus interval (ISI). Variable of interest to show the startle response was maximum velocity (mm/s) with 1 s intervals, since the startle response is a short burst of activity best captured by this parameter. When subjects did not show a clear response to the first stimulus (values lower than 15 mm/s) they were discarded from analysis. Larvae were individually tracked using the DanioVision system and EthoVision XT software (Noldus Information Technology, Wageningen, The Netherlands). This behavioral test was done between 12:00 and 16:00.
Juvenile social interaction: At 30 dpf, an assessment of social contact behavior was carried out using a glass tank (9 cm length × 5 cm height × 7 cm width). Juvenile fish were separated into pairs of the same genotype and transferred to glass tanks with 150 ml water (water depth 3 cm). Each member of the pairs was taken from different home tanks. We tracked the pairs' movements for 6 min, and results included the total duration in proximity (with proximity defined as when the distance between two larvae was ≤ 0.8 cm) Each group had 16 fishes for the data analysis using EthoVision 13 software (Noldus Information Technology, Wageningen, The Netherlands).

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Juvenile shoaling behavior: Zebrafish have the innate tendency of forming tight groups of individuals (shoals), which is considered a form of social interaction that serves many purposes in nature, including foraging, avoiding predation and mating (Pitcher, 1986). The average nearest neighbor distance in a shoal significantly decreases with the age of zebrafish and this change appears particularly robust between 30 and 40 dpf (Buske and Gerlai, 2012).We evaluated this behavior utilizing cohorts of four 40 dpf fish in a round white polyethylene plastic flat-bottomed container (12 cm height, 12 cm diameter) with 350 mL of fish system water (5.0cm depth). Before testing, fish were habituated for 5min followed by video recording for 10 min with a camera at a fixed height (60 cm) from the top of the container. All videos were analyzed with EthoVision XT 13 software, using the default setting (the center-point detection of the unmarked animals). The mean of the interfish distance (defined as distance between the body center of every member of the shoal) was quantified from the average data from all trials (n = 4 trials per genotype). The proximity duration (in seconds) was defined as total duration of time a fish stayed close to the shoal fish (within 1 cm).
Adult social preference: This behavioral test has been described previously (Baronio et al., 2018).
Briefly, the social preference of mao +/+ and mao +/adult animals was evaluated in an acrylic apparatus (29 cm length × 19 cm height × 29 cm width) divided by a transparent wall into two chambers, one of which was subdivided in two smaller compartments. A group of eight fish, serving as stimulus, was placed in one of the compartments, and the other compartment was filled with stones and plant imitations. The tested fish was placed in the other chamber, and the time spent in social, non-social and distal zones was measured with EthoVision 13 software (Noldus Information Technology, Wageningen, The Netherlands).
Adult locomotor activity and thigmotaxis: Adult fish were individually tracked in separate cylindrical observation tanks (inner diameter 22 cm and water depth 8 cm), as described previously (Baronio et al., 2018). The fish had 10 min of habituation time in the tank before the tracking started. The swimming performance of the animals was automatically detected and tracked for 10 min by a digital video camera connected to a standard PC computer system running the Quantification was done by Ct value comparison, using the Ct value of ribosomal protein large subunit 13a (rpl13a) as the reference control.

In situ hybridization
We used 4% paraformaldehyde (PFA)-fixed dissected larval brains and followed the protocol described by Thisse & Thisse with slight modifications to perform the in situ hybridization as described earlier (Thisse and Thisse, 2008). The digoxigenin (DIG) RNA labelling kit (Roche Diagnostics, Germany) was used to produce antisense DIG-labelled RNA probes. The specificity of the probes and clones have been described earlier (Chen et al., 2009;Chen et al., 2016;Kaslin et al., 2004;Peitsaro et al., 2007;Sundvik et al., 2011;Wang et al., 2006). The prehybridization and hybridization steps were performed at 60C. Sheep anti-digoxigenin-AP Fab fragments (1:5000; Roche Diagnostics, Germany) were used to detect the in situ hybridization signals. Staining was performed with nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl-phosphate and samples were incubated at room temperature in the dark. Stained samples were immersed in 80% glycerol, embedded between two cover glasses and analyzed under brightfield optics using a Leica DM IRB inverted microscope.

MAO activity histochemistry
Monoamine oxidase enzyme histochemistry and activity assay was carried out as described (Sallinen et al., 2009b). Briefly, 4% EDAC fixed 40 dpf brain samples were washed 2 x 10 min in PBS and then incubated in 0.05 M Tris-HCl buffer containing 0.08 g/l DAB, 1 g/l tyramine, 1 g/l peroxidase, 6 g/l NiSO4 for 2h at RT.

Microscopy and imaging
Brightfield images were taken with a Leica DM IRB inverted microscope with a DFC 480 charge-coupled device camera. Z-stacks were processed with Leica Application Suite software.
Immunofluorescence samples were examined using a Leica TCS SP2 AOBS confocal microscope.
The Alexa 488-and 568-labelled secondary antibodies were detected using a 488 nm argon laser and a 568 nm diode laser, respectively. Emission was detected at 500-550 nm and 560-620 nm, respectively. Stacks of images taken at 1.0 μm intervals were compiled and the maximum intensity projection algorithm was used to produce final images with Leica Confocal software. Cell numbers were counted in each 1.0 μm optical slice using ImageJ 1.52b software (National Institutes of Health, Bethesda, USA).

Data and statistical analysis
Data shown are representative of a minimum of two independent biological replicates.
Data were analyzed by Student's t-test, one-way analysis of variance (ANOVA) followed by Tukey's post hoc test or two-way multiple comparisons ANOVA followed by Tukey's post hoc

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test, and P < 0.05 was considered statistically significant. Data analysis was performed by GraphPad Prism version 7 software (San Diego, USA). Figure 1 -Behavioral phenotype of mao mutants. (A) mao -/larvae were hypoactive during diurnal and nocturnal (grey shaded area) locomotor activity tracking at 6 dpf. n=16 for mao +/+ , n=16 for mao +/and n=15 for mao -/-. (B) Larvae at 6 dpf were exposed to alternating 2 min periods of light and darkness (grey shaded areas), with three periods of darkness in total. n=15 mao +/+ and n=16 for mao +/and mao -/-(C-E) Analysis of the larval response to sudden darkness in 1 s bins. (F) Mean values of maximum velocity during 10 acoustic/vibrational stimuli with a 20s inter-stimulus interval. n=15 for mao +/+ , n=16 for mao +/and n=12 for mao -/-. Two-way ANOVA followed by Tukey's post hoc test was used for statistical analysis. #p<0.05 (mao +/vs. mao +/+ and mao -/-), *p<0.05 (mao +/+ and mao +/vs. mao -/-), §p<0.05 (mao +/+ vs. mao -/-), € p<0.05 (mao +/vs. mao -/-), ‡ p<0.05 (mao +/vs mao +/+ ).   Ventral views of whole-mount 10 dpf larval brains, anterior to the left, processed for notch1a RNA in situ hybridization (ISH). n=4 for each genotype. (C-F) Bar charts showing results of reverse transcriptase -quantitative PCR (RT-qPCR) assays on larvae of the indicated genotype at 10 dpf. n=4 for each genotype. Data are mean ± SEM. One-way ANOVA followed by Tukey's multiple comparisons test for statistical analysis. *p<0.05, **p<0.01.  Representation of the apparatus used to study the social behavior of adult mao +/+ and mao +/fish and traces of swimming pattern from both genotypes. Fish behavior was evaluated during a 10 min of video recording session in a home-made social interaction apparatus. The apparatus is physically divided into three chambers and digital zones (Distal, Social and Non-social) were stablished with EthoVision 13 software. n=8 for each genotype. (B-E) Bar charts showing results of the time spent in each zone. When compared to their siblings, mao +/adult fish did not differ in the time spent closer to the social stimulus, spent less time in the non-social and more time in the distal zone. When the time spent in both distal zone and object zones summed, these mao +/+ fish show a preference for the area closer to the social stimulus, whereas when the same comparison is done with their mao +/siblings, no preference is detected. Data are mean ± SEM. Student's t-test was used for statistical analysis. *p<0.05.  (I, J) Ventral views of whole-mount adult brains, anterior to the right, processed for serta RNA in situ hybridization (ISH). Serta-positive neurons expressed in the raphe nuclei and hindbrain. Data are mean ± SEM. Student's t-test was used for statistical analysis. *p<0.05.