Serotonin antagonists induce anxiolytic and anxiogenic-like behavior in zebrafish in a receptor-subtype dependent manner

PBB-72054; No of Pages 11 Pharmacology, Biochemistry and Behavior xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Pharmacology, Biochemistry and Behavior journal homepage: www.elsevier.com/locate/pharmbiochembeh

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Magda Nowicki a, Steven Tran b,⁎, Arrujyan Muraleetharan a, Stefan Markovic a, Robert Gerlai a,b

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Article history: Received 10 June 2014 Received in revised form 24 July 2014 Accepted 27 September 2014 Available online xxxx

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Keywords: Zebrafish Anxiety Serotonin antagonist Novel environment Behavior

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Motor function and anxiety-like responses are easily quantifiable in zebrafish, a novel model organism for behavioral pharmacology. Activation of serotonin receptors through the use of selective agonists has been shown to alter anxiety-like behaviors in zebrafish. However, few studies have examined the effect of blockade of specific serotonin receptors. In the current study, we examine the effect of 4 serotonin receptor antagonists selective for 5-HT1A, 5-HT1B/D, 5-HT2, and 5-HT3 receptors on zebrafish motor and anxiety-like responses. Exposure to the receptor antagonists did not change baseline motor responses. However, when placed in a novel environment, zebrafish previously exposed to GR 55562 (5-HT1B/D antagonist) exhibited reduced anxiety-like behavior, whereas zebrafish previously exposed to p-MPPF (5-HT1A antagonist), Ketanserin (5-HT2 antagonist), or Ondasetron (5-HT3 antagonist) exhibited increased anxiety-like behaviors. These results show that drugs developed for mammalian serotonin receptors are efficacious in the zebrafish too, a finding that demonstrates evolutionary conservation of the serotoninergic system. The results also imply that zebrafish may be an appropriate animal model for examining the serotonergic neurotransmitter system in vertebrates. © 2014 Published by Elsevier Inc.

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Serotonergic neurotransmission mediates a large number of behaviors and processes including pain perception (Horjales-Araujo et al., 2013), learning (Izquierdo et al., 2012; Palminteri et al., 2012), aggression (Kulikov et al., 2012), and affect (Williams et al., 2006), including fear and anxiety (Maximino et al., 2013). At least 14 serotonin (5-hydroxytryptamine; 5-HT) receptor subtypes exist in vertebrates and are classified into seven families (5-HT1–5-HT7) based on their downstream effects (Barnes and Neumaier, 2011). All receptors are coupled to G-proteins with the exception of the 5-HT3 receptor. 5-HT1 and 5-HT5 receptors are coupled to inhibitory G proteins which decrease intracellular levels of cAMP by inhibiting adenylyl cyclase. Activation of 5-HT4, 5-HT6 and 5-HT7 receptors increases neuronal transmission by increasing intracellular levels of cAMP (Klee et al., 2012). 5-HT2 receptors are excitatory receptor subtypes which couple to the Gq/G11 signal transduction pathway. Unlike the rest of the serotonin receptor families which are metabotropic, 5-HT3 receptors are ionotropic ligand-gated ion channels. They are composed of five subunits which form a water-filled pore permeable to Na+, Ca2+ and K+. 5-HT3 receptor activation leads to cation influx through the channel, causing depolarization in the post-synaptic terminal. (Walstab et al.,

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Department of Psychology, University of Toronto Mississauga, Canada Department of Cell and Systems Biology, University of Toronto Mississauga, Canada

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Serotonin antagonists induce anxiolytic and anxiogenic-like behavior in zebrafish in a receptor-subtype dependent manner

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⁎ Corresponding author at: Department of Cell and Systems Biology, University of Toronto Mississauga, 3359 Mississauga Road North, Rm 1022D, Mississauga, Ontario L5L 1C6, Canada. Tel.: +1 905 569 4277 (office), +1 905 569 4257 (lab). E-mail address: [email protected] (S. Tran).

2010). The serotonergic system is believed to be highly conserved among vertebrates and may be a good candidate for pharmacological manipulation in a range of species including fish (Lillesaar, 2011; Marston et al., 2011). Zebrafish have extensive homology with mammals at the genetic and neural level, and are rapidly becoming an important model organism for behavioral neuroscience. The serotonergic system in zebrafish has been shown to be physiologically and pharmacologically similar to the mammalian counterpart (Panula et al., 2010; Connors et al., 2014; Maximino et al., in press). Furthermore, genes encoding serotonin receptors in zebrafish show a high nucleotide sequence homology to corresponding human genes (see Klee et al., 2012). Serotonin is important in modulating a number of CNS processes including the function of neural networks that play roles in locomotion and motor function (Brustein et al., 2003; Maximino et al., 2011). For example, serotonin depletion in zebrafish larvae significantly reduces locomotor activity and induces a paralysis-like state (Airhart et al., 2012). Serotoninergic neurotransmission in zebrafish is also important in a number of other behavioral responses including anxiety-like responses (Maximino et al., 2013; Wong et al., 2013). Furthermore, pharmacological activation of serotonin receptors has been shown to reduce behavioral measures of anxiety (Bencan et al., 2009; Connors et al., 2014; Sackerman et al., 2010; Gebauer et al., 2011; Maaswinkel et al., 2012). However, few studies have examined the effects of different serotonin receptor antagonists on motor function or anxiety-like behavioral measures in adult zebrafish.

http://dx.doi.org/10.1016/j.pbb.2014.09.022 0091-3057/© 2014 Published by Elsevier Inc.

Please cite this article as: Nowicki M, et al, Serotonin antagonists induce anxiolytic and anxiogenic-like behavior in zebrafish in a receptor-subtype dependent manner, Pharmacol Biochem Behav (2014), http://dx.doi.org/10.1016/j.pbb.2014.09.022

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Adult zebrafish of the AB strain were bred at the University of Toronto Mississauga Vivarium (Mississauga, Ontario, Canada). The progenitors of this population were originally obtained from the ZFIN Center (Eugene, Oregon, USA). The AB strain was selected for its homozygozity at over 80% of loci (Guryev, 2006), and its frequent use in behavioral neuroscience and mutagenesis studies (Gerlai et al., 2000). Furthermore, the AB strain has been reported to exhibit more robust and quantifiable anxiety-like responses in a novel environment compared to other strains (Sackerman et al., 2010). Zebrafish eggs were collected 2 hours post-fertilization and placed in 2.7 L tanks on a high-density rack system (Aquaneering Inc.). The rack system had multi-stage filtration including a mechanical filter, fluidized glass bed biological filter, active carbon filter, and fluorescent UV light sterilizing unit. Ten percent of the system water (reverse-osmosis deionized water supplemented with 60 mg/L Instant Ocean Sea Salt (Big Al's

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1.2. Experimental design and procedure

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The experiment was intended to characterize the behavioral responses to a range of doses of four serotonin receptor antagonists: p-MPPF, GR 55562, Ketanserin, and Ondansetron, in a novel tank exploration task. On the day of testing, 2.7 L housing tanks were moved to the testing room and animals were allowed to habituate for 30 minutes prior to testing. Individual zebrafish were first exposed to different concentrations of p-MPPF, GR 55562, Ketanserin, and Ondansetron (0.0, 0.1, 0.5, 1.0 mg/L) for 30 minutes in a 1 L tank containing the appropriate drug concentration (n = 16) to examine changes in baseline motor responses. Drug immersion was chosen as the method of delivery over other methods (e.g. injection) because of its less invasive nature and frequent use in assays using adult zebrafish (Levin et al., 2007; Maximino et al., 2013; Sackerman et al., 2010; Connors et al., 2014). For example, drug uptake for citalopram (a serotonin reuptake inhibitor) from the water bath into zebrafish brain tissue confirmed by radioligand binding assays is approximately 1/1000 (Sackerman et al., 2010). The novel tank paradigm used in the current study is sensitive to measures of anxiety and the use of an invasive drug delivery method would have introduced potential confounds. Since the pharmacodynamic and pharmacokinetic properties of these drugs are unknown in zebrafish, the concentrations and duration of exposure chosen for this study are based on unpublished pilot data from our laboratory showing significant reductions in whole brain serotonin and its metabolite (5-hydroxyindolacetic acid) 5-HIAA following 30 minutes of drug administration at the highest dose (Tran et al., unpublished data). Following the 30 minute drug exposure, zebrafish were subsequently placed in a 37 L novel tank (drug-free system water) to quantify anxiety-like behaviors. The back and lateral surfaces, of both the drug exposure and novel tanks, were covered with white corrugated plastic sheets to obscure external cues and to provide a uniform testing environment. Video recordings were made from the front view during both the drug exposure period and novel tank exploration. Recordings commenced immediately after the fish was placed in either tank. Water quality parameters for both the drug exposure and novel tanks matched those of housing tanks.

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1.3. Drug administration

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All chemicals were obtained from Sigma-Aldrich. Stock solutions (5 mg/mL) of the water soluble drugs; p-MPPF, GR 55562, and Ondansetron, were made and were further diluted to obtain the appropriate concentration for the drug exposure tanks (0, 0.1, 0.5, 1.0 mg/L) using system water. Ketanserin is insoluble in water and was dissolved in dimethyl sulfoxide (DMSO) to obtain a stock solution of 5 mg/ml, which was further diluted to the appropriate concentrations for the drug exposure tank (0, 0.1, 0.5, 1.0 mg/L Ketanserin) using system water. A final DMSO concentration in all tanks containing Ketanserin including the Ketanserin control was 0.2% (vol/vol). The technique of immersion based drug delivery with zebrafish using DMSO has been implemented by previous studies. The concentration of DMSO employed in these studies reached 0.4% (vol/vol) and has been shown not to significantly alter the motor responses of zebrafish larvae (Irons et al., 2013; Giacomini et al., 2006) or adults (Connors et al., 2014).

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Aquarium)) was replaced daily. Water quality parameters were maintained at optimal conductivity levels (100–300 microsiemens), temperature (28 °C–30 °C), and pH (6.8–7.2). Upon hatching, larvae were fed Larval AP 100. At 21 days post-fertilization, juvenile fish were fed brine shrimp twice a day. Starting at 30 days post-fertilization, zebrafish were fed brine shrimp and a mixture of flake food (2:1 ratio of tetramin: spirulina). Zebrafish were tested at 8 months of age (approximately 50% males and 50% females).

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Although the serotonergic system in zebrafish may be a potential pharmacological target, only two of the seven 5-HT receptor families (5-HT1 and 5-HT2) have been identified in this species (Norton et al., 2008; Schneider et al., 2012). 5-HT1-like receptors have been examined in zebrafish and 2 genes, ht1aa and ht1ab, have been identified as zebrafish homologs of the mammalian gene that encodes the 5-HT1A receptor. A third zebrafish gene, ht1bd, has been found to encode a receptor similar to the 5-HT1B receptor in humans and 5-HT1D receptor in puffer fish. All three genes show widespread expression in both the larval and adult zebrafish brain, with detected presynaptic autoreceptor and postsynaptic receptor activity (Norton et al., 2008). The second family of receptors recently characterized in zebrafish belongs to the 5-HT2-like family. 5-HT2C receptor cDNA was recently sequenced from the zebrafish, and was determined to be highly homologous to human and mouse sequences (Schneider et al., 2012). The potential role these receptor subtypes may play in anxiety-like behaviors in zebrafish is unclear. The purpose of the current study was to characterize the behavioral function of the above receptor subtypes by using antagonists that are known to specifically target 5-HT1A, 5-HT1B/D, and 5-HT2 receptors. In addition, we also employed an antagonist to target 5-HT3 receptors, the only receptor not coupled to a G-protein. Although the gene encoding 5-HT3 receptor has not been identified in zebrafish, behavioral alterations as a result of drug administration may provide indirect evidence for its existence in this species. To examine the behavioral functions of each serotonin receptor subtype, we first characterize the dose dependent effects of four different serotonin receptor antagonists on zebrafish motor responses: p-MPPF dihydrochloride (5-HT1A antagonist), GR 55562 (5-HT1B/D antagonist), Ketanserin tartrate (5-HT2 antagonist), and Ondansetron hydrochloride (5-HT3 antagonist). We then focus our analyses on behavioral responses exhibited by zebrafish in a novel tank paradigm, a mildly aversive environment, in an attempt to quantify potential effects of these drugs on anxiety (Levin et al., 2007; Bencan and Levin, 2008; Egan et al., 2009). The distinction between fear and anxiety has been blurred in the past; nevertheless, these two phenomena have been distinguished in both the animal and human literature (for a recent review see Gerlai, 2010). For zebrafish, fear versus anxiety has been defined similarly as for mammals (Gerlai, 2010, 2013). Briefly, fear is operationally defined as a set of responses directly induced by the appearance/delivery of specific aversive stimuli, whereas anxiety is defined as responses that do not require the actual appearance or presence of such stimuli. Although serotonergic neurotransmission mediates a large number of behaviors, we restrict our analysis to motor and anxiety related responses which have been well defined and found quantifiable in zebrafish (Tran and Gerlai, 2013; Tran et al., in press; Levin et al., 2007).

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Please cite this article as: Nowicki M, et al, Serotonin antagonists induce anxiolytic and anxiogenic-like behavior in zebrafish in a receptor-subtype dependent manner, Pharmacol Biochem Behav (2014), http://dx.doi.org/10.1016/j.pbb.2014.09.022

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1.4. Quantification of behavior

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1.5. Statistical analysis

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A 2-way repeated measures ANOVA was performed with drug concentration (4 levels) as the between-subject factor, and time (30 one-minute intervals) as the repeated measures factor. In order to compare the effects of drug exposure on zebrafish motor responses and since multiple range post hoc comparison tests are not appropriate for repeated measures, an average of the last 3 minutes of drug exposure was calculated. To quantify changes during novel tank exploration, an average of the first 3 minutes of novel tank exploration was calculated since prior studies have shown significant anxiogenic-like effects during this period (Tran and Gerlai, 2013; Wong et al., 2010). A oneway ANOVA was subsequently conducted with concentration as the between-subjects factor (4 levels). In case significant main effects or interaction terms were found by ANOVA, Tukey's honestly significant difference (HSD) post-hoc tests were subsequently conducted. The analysis for drug exposure and novel tank exploration was conducted separately for each drug. Outliers were removed from the data set using box-plots as recommended by Williamson and Kendrick (1989) based on freezing duration. As a result, trials where zebrafish froze for more than 20% of the time during drug exposure and/or novel tank exposure were removed as outliers (a similar number of outliers were detected for each group). For this reason, a total of 28 trials out of 511 were removed from the analysis.

Zebrafish in the drug exposure tank demonstrated a significant time-dependent increase in distance from bottom, variance of distance to bottom, and total distance traveled, and a time-dependent decrease in absolute angular velocity (Fig. 2; also see Table 1 for details of the results of statistical analyses). Analysis of average performance in the last 3 minutes of the drug exposure session determined no main effect of the drug on any of the behavioral measures suggesting that drug treated zebrafish were not significantly different from controls. When zebrafish were exposed to the novel environment, there was also a significant time-dependent increase in distance from bottom, variance of distance from bottom, and total distance traveled, as well as a time-dependent decrease in absolute angular velocity (Fig. 2). However, analysis of average performance in the first 3 minutes of exposure to the novel tank determined a significant main effect of drug exposure on all behavioral measures. Tukey post-hoc multiple range comparison test showed that control fish had significantly higher angular velocities compared to fish previously exposed to 0.1 mg/L of the drug (p = 0.018). Fish previously exposed to 0.1 mg/L of the drug swam significantly higher in the novel tank compared to control fish (p = 0.027). Fish previously exposed to 0.1 mg/L swam a significantly longer distance than control fish (p = 0.027) during the first 3 minutes of being in the novel environment. Finally, Fish previously exposed to 0.1 mg/L swam a significantly longer distance than control fish (p = 0.027) during the first three minutes of being in the novel environment.

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Video recordings were subsequently replayed and quantified using EthoVision XT 8.0 (Noldus Information Technology, Wageningen, The Netherlands), an automated video-tracking software using dynamic subtraction (a pixel subtraction method). The dependent variables measured by EthoVision included the following: amount of time spent freezing (s), distance from the bottom (cm), total distance traveled (cm), absolute angular velocity (degree/s, a measure of erratic movement, i.e. overall amount and speed of turning), and variance of distance from the bottom (cm 2 , a measure of vertical exploration).

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2. Results

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Zebrafish in the drug exposure tank exhibited a significant timedependent increase in distance from bottom, variance of distance to bottom, and total distance traveled (see Table 1 for details of the results of statistical analyses). In addition, zebrafish also demonstrated a time-dependent decrease in absolute angular velocity (Fig. 1). Repeated measures ANOVA determined a main effect of drug exposure on absolute angular velocity. There was also a significant drug × time interaction on total distance traveled. However, analysis of average performance in the last 3 minutes of the drug exposure session determined no main effect of the drug on any of the behavioral measures suggesting that drug treated zebrafish were not significantly different from controls at this point. When zebrafish were exposed to the novel environment, there was also a time-dependent increase in distance from bottom, variance of distance from bottom, and total distance traveled, as well as a time-dependent decrease in absolute angular velocity (Fig. 1). However, analysis of average performance in the first 3 minutes of exposure to the novel tank determined a significant main effect of drug exposure on absolute angular velocity. Tukey post-hoc multiple range comparison test showed that fish previously treated with 0.1 mg/L of the drug had a higher absolute angular velocity compared to controls and fish previously treated with 1 mg/L (p b 0.05).

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Zebrafish in the drug exposure tank demonstrated a significant time-dependent increase in distance from bottom, variance of distance to bottom, and total distance traveled and a time-dependent decrease in absolute angular velocity (Fig. 3; also see Table 1 for details of the results of statistical analyses). Repeated measures ANOVA determined a significant main effect of drug exposure and drug × time interaction for total distance traveled. Analysis of average performance in the last 3 minutes of drug exposure determined a significant main effect of drug exposure. Tukey post-hoc multiple range comparison test revealed that fish exposed to 0.1 mg/L of ketanserin traveled a significantly larger distance than control fish (p b 0.01). When zebrafish were exposed to the novel environment, there was also a significant time-dependent increase in distance from bottom, variance of distance from bottom, and total distance traveled, as well as a time-dependent decrease in absolute angular velocity (Fig. 3). Analysis of average performance in the first 3 minutes of novel tank exposure determined a significant main effect of drug exposure on absolute angular velocity and distance to bottom. Tukey post-hoc multiple range comparison test showed that control fish had a significantly lower angular velocity compared to fish previously exposed to any concentration of ketanserin (p b 0.05). Control fish swam significantly farther away from the bottom of the tank compared to fish previously exposed to 0.1 mg/L of ketanserin (p = 0.031).

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Zebrafish in the drug exposure tank demonstrated a significant time-dependent increase in distance from bottom, variance of distance to bottom, and total distance traveled (Fig. 4; also see Table 1 for details of the results of statistical analyses). In addition, zebrafish also demonstrated a time-dependent decrease in absolute angular velocity. Analysis of average performance in the last 3 minutes of drug exposure determined that there was no main effect of the drug on any behavioral measures. When zebrafish were exposed to the novel environment, there was also a time-dependent increase in distance from bottom, variance of distance from bottom, and total distance traveled, as well as a time-dependent decrease in absolute angular velocity (Fig. 4). A

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Please cite this article as: Nowicki M, et al, Serotonin antagonists induce anxiolytic and anxiogenic-like behavior in zebrafish in a receptor-subtype dependent manner, Pharmacol Biochem Behav (2014), http://dx.doi.org/10.1016/j.pbb.2014.09.022

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Table 1 Statistical results of repeated measures analyses of variance (ANOVAs) conducted to investigate drug effects and the effect of time (interval) in the novel tank. The last column of the table shows the results of the univariate ANOVAs conducted using data averaged for the last 3 minutes of drug exposure (full drug penetration to the brain is expected), and also for the first 3 minutes of novel tank exposure (highest anxiety response is expected to be induced by the environment). F and p values are shown. Degrees of freedom are also indicated in between brackets.

t1:5

Repeated measures ANOVA

Univariate ANOVA

t1:6

m-PPF(5-HT1A antagonist)

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Behavior

Tank

Time

Drug

Time × drug

Drug

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Absolute angular velocity

Drug exposure

F(29, 1247) = 6.542, p b 0.001 F(29, 1740) = 3.958, p b 0.001 F(29,1247) = 5.763, p b 0.001 F(29, 1740) = 3.231, p b 0.001 F(29, 1247) = 6.044, p b 0.001 F(29, 1740) = 1.966, p b 0.01 F(29, 1247) = 6.171, p b 0.001 F(29, 1740) = 4.032, p b 0.001

F(3, 43) = 3.174, p = 0.034 F(3, 60) = 6.503, p b 0.01 F(3, 43) = 0.313, p = 0.816 F(3, 60) = 0.591, p = 0.623 F(3, 43) = 0.387, p = 0.0763 F(3, 60) = 2.276, p = 0.089 F(3, 43) = 0.099, p = 0.960 F(3, 60) = 0.126, p = 0.944

F(87, 1247) p = 0.997 F(87, 1740) p = 0.801 F(87, 1247) p = 0.999 F(87, 1740) p = 0.929 F(87, 1247) p = 0.681 F(87, 1740) p b 0.001 F(87, 1247) p = 0.521 F(87, 1740) p = 0.608

F(3, 43) = p = 0.218 F(3, 60) = p = 0.011 F(3, 43) = p = 0.751 F(3, 60) = p = 0.656 F(3, 43) = p = 0.878 F(3, 60) = p = 0.293 F(3, 43) = p = 0.806 F(3, 60) = p = 0.989

F(29, 1711) p b 0.001 F(29, 1769) p b 0.001 F(29, 1711) p b 0.001 F(29, 1769) p b 0.001 F(29, 1711) p b 0.001 F(29, 1769) p b 0.001 F(29, 1711) p b 0.001 F(29, 1769) p b 0.001

F(3, 59) = 0.978, p = 0.409 F(3, 61) = 2.458, p = 0.071 F(3, 59) =0.727, p = 0.540 F(3, 61) = 1.402, p = 0.251 F(3, 59) = 0.143, p = 0.934 F(3, 61) = 1.313, p = 0.278 F(3, 59) = 0.472, p = 0.703 F(3, 61) = 1.176, p = 0.326

F(87, 1711) p = 0.997 F(87, 1769) p b 0.001 F(87, 1711) p = 0.037 F(87, 1769) p = 0.115 F(87, 1711) p = 0.885 F(87, 1769) p = 0.609 F(87, 1711) p = 0.141 F(87, 1769) p b 0.001

F(3, 55) = p = 0.402 F(3, 55) = p b 0.01 F(3, 55) = p = 0.620 F(3, 55) = p = 0.224 F(3, 55) = p b 0.01 F(3, 55) = p = 0.865 F(3, 55) = p = 0.173 F(3, 55) = p = 0.694

F(87, 1595) p = 0.989 F(87, 1595) p = 0.518 F(87, 1595) p = 0.919 F(87, 1595) p = 0.025 F(87, 1595) p b 0.001 F(87, 1595) p = 0.483 F(87, 1595) p = 0.599 F(87, 1595) p = 0.101

t1:13

Variance of distance to bottom

Drug exposure

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GR 55562 (5-HT1B/D antagonist) Absolute angular velocity

Drug exposure

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Novel tank Distance to bottom

Drug exposure

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Novel tank Total distance traveled

Drug exposure

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Novel tank Variance of distance to bottom

Drug exposure

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Ketanserin (5-HT2 antagonist) Absolute angular velocity

Drug exposure

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Novel tank Distance to bottom

Drug exposure

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Total distance traveled

Drug exposure

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Novel tank

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Novel tank Variance of distance to bottom

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Ondansetron (5-HT3 antagonist) Absolute angular velocity

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Distance to bottom

t1:41 t1:42

t1:45

Drug exposure Novel tank Drug exposure Novel tank

Total distance traveled

t1:43 t1:44

Drug exposure

Novel tank

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Variance of distance to bottom

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= 6.326, = 9.053, = 4.664, = 9.207, = 7.463, = 9.554, = 8.720,

= 13.728,

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F(29, 1595) p b 0.001 F(29, 1595) p b 0.001 F(29, 1595) p b 0.001 F(29, 1595) p b 0.001 F(29, 1595) p b 0.001 F(29, 1595) p b 0.001 F(29, 1595) p b 0.001 F(29, 1595) p b 0.001 F(29, 1595) p b 0.001 F(29, 1711) p b 0.001 F(29, 1595) p b 0.001 F(29, 1711) p b 0.001 F(29, 1595) p b 0.001 F(29, 1711) p b 0.001 F(29, 1595) p b 0.001 F(29, 1711) p b 0.001

= 5.485, = 5.779, = 7.504, = 8.765, = 12.382, = 4.353, = 15.366, = 10.728,

= 23.970, = 8.913, = 16.979, = 4.343, = 24.317, = 6.689, = 19.043, = 9.469,

F(3, 55) = p = 0.597 F(3, 59) = p = 0.018 F(3, 55) = p = 0.292 F(3, 59) = p = 0.884 F(3, 55) = p = 0.407 F(3, 59) = p = 0.590 F(3, 55) = p = 0.138 F(3, 59) = p = 0.998

0.995, 4.643, 0.596, 1.504, 4.323, 0.244, 1.722, 0.485,

0.633, 3.636, 1.272, 0.218, 0.985, 0.644, 1.912, 0.012,

= 0.868, = 0.582,

F

= 0.783, = 0.921,

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t1:11

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t1:10

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t1:9

= 0.630,

F(87, 1595) p = 0.601 F(87, 1711) p b 0.001 F(87, 1595) p = 0.992 F(87, 1711) p = 0.992 F(87, 1595) p = 0.887 F(87, 1711) p = 0.926 F(87, 1595) p = 0.493 F(87, 1711) p = 0.521

= 1.737, = 0.985,

= 0.951,

= 0.622, = 2.192, = 1.298, = 1.191, = 0.819, = 0.950, = 1.169, = 1.646,

= 0.682, = 0.986, = 0.792, = 1.330, = 2.061, = 0.999, = 0.954, = 1.204,

= 0.954, = 2.276, = 0.664, = 0.664, = 0.817, = 0.786, = 0.812, = 0.985,

F(3, 59) = p = 0.089 F(3, 61) = p = 0.028 F(3, 59) = p = 0.278 F(3, 61) = p = 0.048 F(3, 59) = p = 0.902 F(3, 61) = p = 0.048 F(3, 59) = p = 0.329 F(3, 61) = p = 0.027

F(3, 55) = p = 0.841 F(3, 55) = p b 0.01 F(3, 55) = p = 0.744 F(3, 55) = p = 0.028 F(3, 55) = p = 0.012 F(3, 55) = p = 0.706 F(3, 55) = p = 0.740 F(3, 55) = p = 0.083

F(3, 55) = p = 0.348 F(3, 59) = p b 0.01 F(3, 55) = p = 0.792 F(3, 59) = p = 0.710 F(3, 55) = p = 0.084 F(3, 59) = p = 0.417 F(3, 55) = p = 0.464 F(3, 59) = p = 0.908

1.540, 4.057, 0.404, 0.541, 0.226, 1.268, 0.326, 0.041,

2.280, 3.230, 1.317, 2.786, 0.191, 2.789, 1.168, 3.285,

0.277, 6.326, 0.413, 3.259, 3.984, 0.468, 0.419, 2.345,

1.122, 5.809, 0.346, 0.462, 2.335, 0.962, 0.867, 0.183,

Please cite this article as: Nowicki M, et al, Serotonin antagonists induce anxiolytic and anxiogenic-like behavior in zebrafish in a receptor-subtype dependent manner, Pharmacol Biochem Behav (2014), http://dx.doi.org/10.1016/j.pbb.2014.09.022

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Fig. 1. The time dependent behavioral changes in response to acute exposure to m-PPF (5-HT1A antagonist) are shown for 1 minute intervals for absolute angular velocity (drug exposure tank panel A; novel aquarium panel B), distance to bottom (drug exposure tank panel D; novel aquarium panel E), total distance traveled (drug exposure tank panel E; novel aquarium panel F) and variance of distance to bottom (drug exposure tank panel G; novel aquarium panel H). Mean ± S.E.M. is shown. The average of the last 3 minutes in the drug exposure tank (full penetration of drug is expected) is used to compare different concentration groups using Tukey post-hoc HSD tests, shown as bar graphs, insets of panels A, C, E, and G. The average of the first 3 minutes of novel tank exposure (strongest anxiety induced by novelty) is used to compare different concentration groups using Tukey post-hoc HSD tests, shown as bar graphs, insets of panels B, D, F, and H. Significant (p b 0.05) differences among groups are indicated by the small letters above the bars. Bars that do not share the same letter are significantly different. Bars with no letters are not significantly different. For details of the results of statistical analyses see Results.

Please cite this article as: Nowicki M, et al, Serotonin antagonists induce anxiolytic and anxiogenic-like behavior in zebrafish in a receptor-subtype dependent manner, Pharmacol Biochem Behav (2014), http://dx.doi.org/10.1016/j.pbb.2014.09.022

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Fig. 2. The time dependent behavioral changes in response to acute exposure to GR 55562 (5-HT1B/D antagonist) are shown for 1 minute intervals for absolute angular velocity (drug exposure tank panel A; novel aquarium panel B), distance to bottom (drug exposure tank panel D; novel aquarium panel E), total distance traveled (drug exposure tank panel E; novel aquarium panel F) and variance of distance to bottom (drug exposure tank panel G; novel aquarium panel H). Mean ± S.E.M. is shown. The average of the last 3 minutes in the drug exposure tank (full penetration of drug is expected) is used to compare different concentration groups using Tukey post-hoc HSD tests, shown as bar graphs, insets of panels A, C, E, and G. The average of the first 3 minutes of novel tank exposure (strongest anxiety induced by novelty) is used to compare different concentration groups using Tukey post-hoc HSD tests, shown as bar graphs, insets of panels B, D, F, and H. Significant (p b 0.05) differences among groups are indicated by the small letters above the bars. Bars that do not share the same letter are significantly different. Bars with no letters are not significantly different. For details of the results of statistical analyses see Results.

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repeated measures ANOVA determined a significant main effect of drug exposure and drug × time interaction on absolute angular velocity. Analysis of average angular velocity in the first 3 minutes of the novel tank determined a main effect of drug exposure. Tukey post-hoc multiple

range comparison test revealed that fish previously exposed to 1.0 mg/L of ondansetron exhibited higher angular velocity compared to controls and also compared to fish exposed to 0.1 mg/L and 0.5 mg/L of the drug (p b 0.05).

Please cite this article as: Nowicki M, et al, Serotonin antagonists induce anxiolytic and anxiogenic-like behavior in zebrafish in a receptor-subtype dependent manner, Pharmacol Biochem Behav (2014), http://dx.doi.org/10.1016/j.pbb.2014.09.022

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Fig. 3. The time dependent behavioral changes in response to acute exposure to Ketanserin (5-HT2 antagonist) are shown for 1 minute intervals for absolute angular velocity (drug exposure tank panel A; novel aquarium panel B), distance to bottom (drug exposure tank panel D; novel aquarium panel E), total distance traveled (drug exposure tank panel E; novel aquarium panel F) and variance of distance to bottom (drug exposure tank panel G; novel aquarium panel H). Mean ± S.E.M. is shown. The average of the last 3 minutes in the drug exposure tank (full penetration of drug is expected) is used to compare different concentration groups using Tukey post-hoc HSD tests, shown as bar graphs, insets of panels A, C, E, and G. The average of the first 3 minutes of novel tank exposure (strongest anxiety induced by novelty) is used to compare different concentration groups using Tukey post-hoc HSD tests, shown as bar graphs, insets of panels B, D, F, and H. Significant (p b 0.05) differences among groups are indicated by the small letters above the bars. Bars that do not share the same letter are significantly different. Bars with no letters are not significantly different. For details of the results of statistical analyses see Results.

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environment. The diving response has been used to quantify anxietylike behaviors in small molecule screens (Levin et al., 2007; Bencan and Levin, 2008; Egan et al., 2009). In the current study, we characterize novelty induced behavioral responses in detail by analyzing the

Please cite this article as: Nowicki M, et al, Serotonin antagonists induce anxiolytic and anxiogenic-like behavior in zebrafish in a receptor-subtype dependent manner, Pharmacol Biochem Behav (2014), http://dx.doi.org/10.1016/j.pbb.2014.09.022

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Fig. 4. The time dependent behavioral changes in response to acute exposure to Ondansetron (5-HT3 antagonist) are shown for 1 minute intervals for absolute angular velocity (drug exposure tank panel A; novel aquarium panel B), distance to bottom (drug exposure tank panel D; novel aquarium panel E), total distance traveled (drug exposure tank panel E; novel aquarium panel F) and variance of distance to bottom (drug exposure tank panel G; novel aquarium panel H). Mean ± S.E.M. is shown. The average of the last 3 minutes in the drug exposure tank (full penetration of drug is expected) is used to compare different concentration groups using Tukey post-hoc HSD tests, shown as bar graphs, insets of panels A, C, E, and G. The average of the first 3 minutes of novel tank exposure (strongest anxiety induced by novelty) is used to compare different concentration groups using Tukey post-hoc HSD tests, shown as bar graphs, insets of panels B, D, F, and H. Significant (p b 0.05) differences among groups are indicated by the small letters above the bars. Bars that do not share the same letter are significantly different. Bars with no letters are not significantly different. For details of the results of statistical analyses see Results.

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temporal trajectories of behavior, and report on four behavioral measures that reflect novelty-induced anxiety-like responses. Several of these measures have previously been reported including an initial

preference for the bottom of the tank (Levin et al., 2007; Wong et al., 341 2010; Sackerman et al., 2010; Maximino et al., 2013), decreased total 342 distance traveled (Levin et al., 2007; Bencan and Levin, 2008) and 343

Please cite this article as: Nowicki M, et al, Serotonin antagonists induce anxiolytic and anxiogenic-like behavior in zebrafish in a receptor-subtype dependent manner, Pharmacol Biochem Behav (2014), http://dx.doi.org/10.1016/j.pbb.2014.09.022

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anxiolytic-like effects of p-MPPF and GR 55562 may result from the initial blockade of post-synaptic 5-HT1A-like and 5-HT1B/D receptors at the lowest concentration in zebrafish. However, at higher concentrations, blockade of pre-synaptic autoreceptors may facilitate serotonergic neurotransmission through disinhibition to diminish the effect of postsynaptic blockade. The 5-HT2-like receptors have been shown to modulate levels of anxiety in rodents (Gibson et al., 1994; Nunes-de-Souza et al., 2008; Dhonnchadha et al., 2003). In zebrafish, a single gene encoding the 5HT2C receptor has been identified (Schneider et al., 2012). Activation of 5-HT 2C receptors in rodents has been shown to induce both anxiogenic and anxiolytic-like effects depending on the neuroanatomical localization of these receptors (Nunes-de-Souza et al., 2008) and the behavioral paradigms used to quantify behavior (Dhonnchadha et al., 2003). Ketanserin is a selective 5-HT2A/C antagonist in mammals and was shown to induce anxiogenic-like effects in rats, whereas 5-HT2A antagonism alone had no effect (Dhonnchadha et al., 2003). In line with prior empirical evidence, we report that exposure to Ketanserin induced anxiogenic-like effects in zebrafish during the novel tank exploration task demonstrated by decreased distance to bottom (increased bottom dwelling) and decreased variance of distance to bottom (decreased vertical exploration), as well as by increased angular velocity (increased erratic movement). Notably, the lowest dose of Ketanserin (0.1 mg/L) was the only drug condition to alter motor patterns during drug exposure. It increased total distance traveled. Ketanserin is a selective 5-HT2 receptor antagonist for 5-HT 2A and 5-HT 2C (Creed-Carson et al., 2011). In addition to these target receptors, it also antagonizes histamine H1 and α1-adrenergic receptors (Van der Starre and Reneman, 1998; Beele et al., 1990). The gene encoding the H1 receptor has been identified in zebrafish and H1 receptor antagonism in zebrafish larvae has been shown to decrease locomotor activity (Peitsaro et al., 2007), similar to the reduction in H1 receptor knockouts in mice (Inoue et al., 1996). Prazosin, a α1-adrenergic receptor antagonist, has also been shown to induce anxiolytic effects by preventing sleep deprivation-induced anxiety in adult zebrafish (Singh et al., 2013). Although the psychopharmacological profile of ketanserin in zebrafish appears to contradict the literature, it may represent a complex interaction between the antagonsism of serotonergic, adrenergic and histaminergic receptors. In the current study, Ketanserin was the only water insoluble drug which required the use DMSO as a vehicle. Control fish in the Ketanserin group exposed to DMSO appeared to have a slightly attenuated anxiety response in the first 3 minutes of novel tank exposure (i.e. lower absolute angular velocity and higher distance to bottom). Prior studies have shown that zebrafish exposed to DMSO exhibit robust reduction of anxiety-like behaviors in the light/dark plus maze as demonstrated by increased total arm entries and total time spent in the white arms (Sackerman et al., 2010). In the current study, the use of DMSO as a vehicle appears to have decreased anxiety-like measures in the novel tank which may have enhanced the detection of ketanserin's anxiogenic effect. Although the use of DMSO as a vehicle may enhance the detection power of behavioral drug screens for anxiogenic compounds by altering the base-line of control fish, this organic solvent should be used with caution when screening for anxiolytic compounds (Sackerman et al., 2010). Since the effects of DMSO are concentration dependent, using lower concentrations without overt behavioral effects may alleviate this concern. Ondansetron exhibits mild anxiolytic properties in certain mammalian species including rodents (Garibova et al., 1999; de Oliveira Cito et al., 2012) and humans (see Freeman et al., 1997), but certain studies have failed to detect significant behavioral alterations (Rodgers et al., 1997). In the current study, we found that exposure to Ondansetron at the highest concentration (1 mg/L) increased angular velocity during novel tank exposure compared to controls, a response we interpret as increased anxiety-like behavior. These results contradict

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decreased variance of distance from bottom, with the latter being interpreted as decreased vertical exploration (Tran and Gerlai, 2013). In addition, we also report an initially high absolute angular velocity during the diving response, which has been associated with and is now regarded as a good alternative measure of erratic movement (Tran et al., in press). Although it may be difficult to speculate whether this initially high absolute angular velocity represents a “diving response” or general erratic movement, it is likely a good measure of anxiogenic-like behavior as it is reduced by anxiolytic drugs. Exposure to different serotonin receptor antagonists in the current study was shown not to alter any of these motor responses under the majority of the drug doses. However, when drug exposed fish were subsequently tested in a novel environment, the initial diving response was differentially altered suggestive of an anxiolytic and anxiogenic effect. For the first time, we show that serotonin receptor antagonism differentially alters anxiety-like behavior in adult zebrafish in the novel tank diving test in a receptor-subtype dependent manner, an effect which appears to be independent of motor impairment. Two genes (ht1aa and ht1ab) encoding 5-HT1A-like receptors have been identified in zebrafish (Norton et al., 2008). Blocking 5-HT1A receptors using p-MPPF (a selective 5-HT1A receptor antagonist) induced anxiogenic-like effects during novel tank exploration: we found it to increase angular velocity (reflective of increased erratic movements). Our finding fits well with the mammalian literature. For example, 5-HT1A receptor knockout mice have been shown to exhibit increased levels of anxiety in an open field task and elevated plus maze (Heisler et al., 1998; Zhuang et al., 1999). Furthermore, activation of 5-HT1A receptors using selective agonists has been shown to reduce anxiety-like measures in infant rats (Brunelli et al., 2009) and in zebrafish (Connors et al., 2014; Bencan et al., 2009; Maximino et al., 2013). A single gene (ht1bd) encoding a receptor similar to the human 5-HT1B and puffer fish 5-HT1D was also identified in zebrafish (Schneider et al., 2012). Blocking 5-HT1B/D receptors using GR 55562 (a 5-HT1B/D receptor antagonist) had anxiolytic effects during novel tank exploration as demonstrated by decreased absolute angular velocity (reduced erratic movement), increased distance to bottom (decreased bottom dwelling), increased variance of distance from bottom (increased vertical exploration), and elevated total distance traveled (increased locomotor activity) in the first 3 minutes. Our findings are similar to those showing an anxiolytic effect of a 5-HT1B antagonist in zebrafish (Maximino et al., 2013) and are also in line with the known behavioral functions of 5-HT1B receptors demonstrated in mammals. For example, 5-HT1B receptor knockout mice have been shown to be less anxious compared to wild-type controls (Zhuang et al., 1999). Activation of 5-HT1B receptors using a selective agonist increases measures of anxiety in the elevated plus maze in rats (Lin and Parsons, 2002). Furthermore, selective 5-HT1B/D agonists have been shown to increase autonomic and subjective measures of anxiety in humans (Amital et al., 2005; de Rezende et al., 2013). Notably, the anxiogenic and anxiolytic effect of p-MPPF and GR 55562, respectively, was only significant at the lowest dose, which is in line with the differential activation of pre- and postsynaptic 5HT1-like receptors (Akimova et al., 2009; Hopwood and Stamford, 2001). For example, p-MPPF has been shown to block postsynaptic receptors at low concentrations and presynaptic 5-HT1A somatodendritic autoreceptors at higher concentrations (Thielen et al., 1996). 5HT1A, 5HT1B, and 5HT1D have been shown to be differentially localized to pre and postsynaptic receptors in mammals (Akimova et al., 2009; McDevitt et al., 2011; Hopwood and Stamford, 2001). Furthermore, autoreceptor activity has been detected for all three 5-HT1-like receptors in the adult and larval zebrafish brain (Norton et al., 2008). Postsynaptic receptors relay serotonin activity to target neurons and propagate signaling. Activation of presynaptic receptors mediates negative feedback by inhibiting the release and synthesis of serotonin (see McDevitt and Neumaier, 2011). The observed anxiogenic and

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Airhart MJ, Lee DH, Wilson TD, Miller BE, Miller MN, Skalko RG, et al. Adverse effects of serotonin depletion in developing zebrafish. Neurotoxicol Teratol 2012;34:152–60. Akimova E, Lanzenberger R, Kasper S. The serotonin-1A receptor anxiety in anxiety disorders. Biol Psychiatry 2009;66:627–35. Amital D, Fostick L, Sasson Y, Kindler S, Amital H, Zohar J. Anxiogenic effects of Sumatriptan in panic disorder: a double-blind, placebo-controlled study. Eur Neuropsychopharmacol 2005;15:279–82. Barnes NM, Neumaier JF. Neuronal 5-HT receptors and SERT. Tocris bioscience scientific review series, 34; 2011. p. 1–15. Beele H, Thierens H, de Ridder L. Direct effects of serotonin, and 5-HT2, α1 and H1 receptor-antagonists on embryonic chick skin in vitro: a morphological and functional study. Cell Biol Int Rep 1990;14:737–46. Bencan Z, Levin ED. The role of α7 and α4β2 nicotinic receptors in the nicotine-induced anxiolytic effect in zebrafish. Physiol. Behav. 2008;95:408–12. Bencan Z, Sledge D, Levin ED. Buspirone, chlordiazepoxide and diazepam effects in a zebrafish model of anxiety. Pharmacol. Biochem. Behav. 2009;94:75–80. Blaser RE, Goldsteinholm K. Depth preference in zebrafish, Danio rerio, control by surface and substrate cues. Anim Behav 2012;83:953–9. Brunelli SA, Aviles JA, Gannon KS, Branscomb A, Shacham S. PRX-00023, a selective serotonin 1A receptor agonist, reduces ultrasonic vocalizations in infant rats bred for high infantile anxiety. Pharmacol. Biochem. Behav. 2009;94:8–15. Connors KA, Valenti TW, Lawless K, Sackerman J, Onaivi ES, Brooks BW, et al. Similar anxiolytic effects of agonists targeting serotonin 5-HT1A or cannabinoid CB receptors on zebrafish behavioural in novel environments. Aquat Toxicol 2014;2014(151): 105–13. Creed-Carson M, Oraha A, Nobrega JN. Effects of 5-HT(2A) and 5-HT(2C) receptor antagonists on acute and chronic dyskinetic effects induced by haloperidol in rats. Behav Brain Res 2011;219:273-27. De Oliveira Cito Mdo C, da Silva FC, Silva MI, Moura BA, Macedo DS, Woods DJ, et al. Reversal of cocaine withdrawal-induced anxiety by ondansetron, buspirone and propranolol. Behav Brain Res 2012;231:116–23. De Rezende MG, Garcia-Leal C, Graeff FG, Del-Ben CM. The 5-HT1D/1B receptor agonist sumatriptan enhances fear of simulated speaking and reduces plasma levels of prolactin. J Psychopharmacol 2013;27:1124–33. Dhonnchadha BAN, Bourin M, Hascoet M. Anxiolytic-like effects of 5-HT2 ligands on three mouse models of anxiety. Behav Brain Res 2003;140:203–14. Egan RJ, Bergner CL, Hart PC, Cachat J, Canavelo PR, Elegante MF, et al. Understanding behavioural and physiolotical phenotypes of stress and anxiety in zebrafish. Behav Brain Res 2009;205:38–44. Freeman III AM, Westphal JR, Norris GT, Roggero BA, Webb PB, Freeman KL, et al. Efficacy of ondansetron in the treatment of generalized anxiety disorder. Depress Anxiety 1997;5:140–1. Garibova TL, Voronina TA, Kraineva VA, Val'dman EA. The anxiolytic effect of ondansetron and its capacity to eliminate the benzodiazepine abstinence syndrome in rats. Eksp Klin Farmakol 1999;62:16–9. Gebauer DL, Pagnussat N, Piato AL, Schaefer IC, Bonan CD, Lara DR. Effects of anxiolytics in zebrafish: similarities and differences between benzodiazepines, buspirone and ethanol. Pharmacol Biochem Behav 2011;99:480–6. Gerlai R. Zebrafish antipredatory responses: a future for translational research? Behav Brain Res 2010;207:223–31. Gerlai R. Antipredatory behaviour of zebrafish: adaptive function and a tool for translational research. Evol. Psychol. 2013;11:591–605. 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. Pharmacol. Biochem. Behav. 2000; 67:773–82. Gerlai R, Fernandes Y, Pereira T. Zebrafish (Danio rerio) responds to the animated image of a predator: towards the development of an automated aversive task. Behav Brain Res 2011;201:318–24. Gibson EL, Barnfield AMC, Curzon G. Evidence that mCPP-induced anxiety in the plusmaze is mediated by postsynaptic 5-HT2c receptors but not by sympathomimetic effects. Neuropharmacology 1994;33:457–65. Heisler LK, Chu HM, Brennan TJ, Danao JA, Bajwa P, Parsons LH, et al. Elevated anxiety and antidepressant-like responses in serotonin 5-HTA receptor mutant mice. Proc Natl Acad Sci U S A 1998;95. [15049-15049]. Herculano AM, Maximino C. Serotonergic modulation of zebrafish behavior: towards a paradox. Prog. Neuro-Psychopharmacol. Biol. Psychiatry 2014. [in press]. Hopwood SE, Stamford JA. Multiple 5-HT1 autoreceptor subtypes govern serotonin release in dorsal and median raphe nuclei. Neuropharmacology 2001;40: 508–19. Horjales-Araujo E, Demontis D, Lund EK, Vase L, Finnerup NB, Borglum AD, et al. Emotional modulation of muscle pain is associated with polymorphisms in the serotonin transporter gene. Pain 2013;154:1469–76. Inoue I, Yanai K, Kitamura D, Taniuchi I, Kobayashi T, Niimura K, et al. Impaired locomotor activity and exploratory behaviour in mice lacking histamine H1 receptors. Proc Natl Acad Sci U S A 1996;93:13316–20. Izquierdo A, Carlos K, Ostrander S, Rodriguez D, McCall-Craddolph A, Yagnik G, et al. Impaired reward learning and intact motivation after serotonin depletion in rats. Behav Brain Res 2012;233:494–9. Klee EW, Schneider H, Clark KJ, Cousin MA, Ebbert JO, Hooten WM, et al. Zebrafish: a model for the study of addiction genetics. Hum Genet 2012;131:977–1008. Kulikov AV, Osipova DV, Naumenko VS, Terenina E, Mormede P, Popova NK. A pharmacological evidence of positive association between mouse intermale aggression and brain serotonin metabolism. Behav Brain Res 2012;233:113–9. Levin ED, Bencan Z, Cerutti DT. Anxiolytic effects of nicotine in zebrafish. Physiol. Behav. 2007;90:54–8. Lillesaar C. The serotonin system in fish. J Chem Neuroanat 2011;41:294–308.

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the current literature. Notably, 5-HT3 genes have not yet been cloned from the zebrafish and Ondansetron's target binding profile once 478 thoroughly examined in the zebrafish may include unexpected re479 ceptors. Nevertheless, it should be noted that the htr3a gene has 480 been predicted and annotated in the zebrafish genome (Ensemble 481 ZV-9: ENSDARG00000080012 www.ensembl.org/Danio_rerio/Info/ 482 Index) and thus cloning 5-HT3 gene(s) is a likely possibility. Once 483 cloned, analyses of these genes and their protein products along with 484 radioligand binding essays may provide insights about the specificity 485 of Ondansetron. Irrespective of the current uncertainties, our results 486 imply that this drug does bind and functionally affect some molecular 487 targets in the zebrafish brain. 488 All anxiogenic drugs tested in this study altered the novelty induced 489 anxiety-like response by increasing absolute angular velocity (erratic 490 turning movement), while the anxiolytic drug decreased absolute angu491 lar velocity. However, the remaining measures of anxiety-like behavior 492 (i.e. total distance traveled, distance from bottom, and variance of dis493 tance from bottom) varied depending on the type of drug used. The 494 results suggest that changes in angular velocity during the first three 495 minutes of novel tank exploration may provide the most robust and 496 sensitive measure of anxiety-like behavior, a conclusion that has been 497 already drawn in other studies of the behavior of adult zebrafish Q12498 Q11 (Ahmed et al., 2011; Gerlai et al., 2011; Tran and Gerlai, 2013). 499 It should also be noted that serotonin has been hypothesized to play 500 Q13 a “dual role” in modulating different types of anxiety (Maxminio et al., 501 2012; Maximino et al., 2013; Herculano and Maximino, in press). For 502 example, anxiety-like behavioral measures in the novel tank diving 503 tests correlate negatively with extracellular brain levels of serotonin in 504 zebrafish, whereas anxiety-like measures in the light/dark preference 505 tests correlate positively (Maximino et al., 2013). These contradictory 506 findings published in the literature may be due to the fact that 507 zebrafish's fear and anxiety responses, i.e. their antipredatory behav508 ioral repertoire, is/are highly complex and context (stimulus and 509 environmental condition) dependent (Luca and Gerlai, 2012). An510 other notable possibility is that serotoninergic neurotransmission 511 plays different roles in a neuroanatomical locale dependent manner. 512 Future studies will thus need to examine such potential brain area 513 specific functions along with context specificity perhaps using targeted 514 local manipulation of the serotoninergic system and/or higher resolu515 tion anatomical mapping and functional analysis of this neurotransmit516 ter system under different stimulus conditions. 517 Irrespective of these unresolved questions, our results suggest that 518 the serotonergic system is evolutionarily conserved in the zebrafish 519 with receptors amenable to psychopharmacological analysis. We 520 conclude that adult zebrafish exhibit a rich repertoire of quantifiable 521 behavioral responses that may be pharmacologically altered using 522 drugs developed for mammalian receptors making the zebrafish a 523 useful animal model for high throughput screening of anxiogenic/ 524 anxiolytic compounds in particular and drug discovery in general.

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This study was supported by an NSERC Discovery grant (311637) issued to R.G. and an NSERC USRA issued to M.N.

532

References

533 534

Ahmed O, Seguin D, Gerlai R. An automated predator avoidance task in zebrafish. Behav Brain Res 2011;216:166–71.

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Peitsaro N, Sundvik M, Anichtchik OV, Kaslin J, Panula P. Identification of zebrafish histamine H1, H2, and H3 receptors and effects of histaminergic ligands on behaviour. Biochem Pharmacol 2007;73:1205–14. Rodgers RJ, Cutler MG, Jackson JE. Behavioural effects of mice subchronic buspirone, ondansetron and tianeptine. II. The elevated plus-maze. Pharmacol Biochem Behav 1997;56:295–300. Sackerman J, Donegan JJ, Cunningham CS, Nguyen NN, Lawless K, Long A, et al. Zebrafish behavior in novel environments: effects of acute exposure to anxiolytic compounds and choice of Danio rerio line. Int. J. Comp. Psychol. 2010;23:43–61. Schneider H, Fritzky L, Williams J, Heumann C, Yochum M, Pattar K, et al. Cloning and expression of a zebrafish 5-HT2C receptor gene. Gene 2012;502:108–17. Serotonin patterns locomotor network activity in the developing zebrafish by modulating quiescent periods. Serotonin patterns locomotor network activity in the developing zebrafish by modulating quiescent periods. J Neurobiol 2003;57:303–22. Singh A, Subhashini N, Sharma S, Mallick BN. Involvement of the α1-adrenoceptor in sleep-waking and sleep loss-induced anxiety behaviour in zebrafish. Neuroscience 2013;245:136–47. Thielen RJ, Fangon NB, Frazer A. 4-(2′-Methoxyphenyl)-1-[2′-[N-(2″-pyridinyl)-piodobenzamido]ethyl] piperazine and 4-(2′-methoxyphenyl)-1-[2′-[N-(2″-pyridinyl)p-fluorobenzamido]ethyl]piperazine, two new antagonists at pre- and postsynaptic serotonin-1A receptors. J Pharmacol Exp Ther 1996;277:661–70. Tran S, Gerlai R. Time-course of behavioural changes induced by ethanol in zebrafish (Danio rerio). Behav Brain Res 2013;252:204–13. Tran S, Chatterjee D, Gerlai R. An integrative analysis of ethanol tolerance and withdrawal in zebrafish (Danio rerio). Behav Brain Res 2014. [in press]. Van der Starre PJ, Reneman RS. The alpha-adrenergic receptor blocking effect of ketanserin and the interaction between alpha-adrenergic and S2-serotonergic receptor blockade. J. Cardiovasc. Pharmacol. 1998;11:54–61. Walstab J, Rappold G, Niesler B. 5-HT(3) receptors: role in disease and target of drugs. Pharmacol Ther 2010;128:146–69. Williams E, Stewart-Knox B, Helander A, McConville C, Bradbury I, Rowland I. Associations between whole-blood serotonin and subjective mood in healthy male volunteers. Biol Psychol 2006;71:171–4. Williamson DF, Kendrick JS. The box plot: a simple visual method to interpret data. Acad. Clin. 1989;110:916–21. Wong K, Elegante M, Bartels B, Elkhayat S, Tien D, Roy S, et al. Analyzing habituation responses to novelty in zebrafish (Danio rerio). Behav Brain Res 2010;208:450–7. Wong RY, Oxendine SE, Godwin J. Behavioural and neurogenomic transcriptome changes in wild-derived zebrafish with fluoxetine treatment. BMC Genomics 2013;14:348. Zhuang X, Gross C, Santarelli L, Compan V, Trillat AC, Hen R. Altered emotional states in knockout mice lacking 5-HT1A or 5-HT1B receptors. Neuropsychopharmacology 1999;21:52–60.

E

Lin D, Parsons LH. Anxiogenic-like effect of serotonin1B receptor stimulation in the rat elevated plus-maze. Pharmacol. Biochem. Behav. 2002;71:581–7. Luca R, Gerlai R. In search of optimal fear inducing stimuli: differential behavioral responses to computer animated images in zebrafish. Behav Brain Res 2012;226: 66–76. Maaswinkel H, Zhu L, Weng W. The immediate and the delayed effects of buspirone on zebrafish (Danio rerio) in an open field test: a 3-D approach. Behav Brain Res 2012; 234:365–74. Marston OJ, Garfield AS, Lora KH. Role o central serotonin and melanocortin systems in the control of energy balance. Eur J Pharmacol 2011;660:70–9. Maximino C, Araujo J, Leao LK, Grisolia AB, Oliveira KR, Lima MG, et al. Possible role of serotonergic system in the neurobehavioural impairment induced by acute methylmercury exposure in zebrafish (Danio rerio). Neurotoxicol Teratol 2011;33:727–34. Maximino C, Puty B, Benzecry R, Araujo J, Lima MG, de Jesus Oliveira Batista E, et al. Role of serotonin in zebrafish (Danio rerio) anxiety: relationship with serotonin levels and effect of buspirone, WAY 100635, SB 224289, fluoxetine and parachlorophenylalanine (pCPA) in two behavioural models. Neuropharmacology 2013;71:83–97. Maximino C, Lima MG, Costa CC, Guedes IM, Herculano AM. Fluoxetine and WAY 100,635 dissociate increases in scototaxis and analgesia induced by conspecific alarm substance in zebrafish (Danio rerio Hamilton 1822). Pharmacol Biochem Behav 2014. [in press]. Maxminio C, Benzecry R, Oliveira KRM, Batista EdJO, Herculano AM, Rosemberg DB, et al. A comparison of the light/dark and novel tank tests in zebrafish. Behaviour 2012;149: 1099–123. McDevitt RA, Neumaier JF. Regulation of dorsal raphe nucleus function by serotonin autoreceptors: a behavioural perspective. J Chem Neuroanat 2011;41:234–46. McDevitt RA, Hiroi R, Mackenzie SM, Robin NC, Cohn A, Kim JJ, et al. Serotonin 1B autoreceptors originating in the caudal dorsal raphe nucleus reduce expression of fear and depression-like behaviour. Biol Psychiatry 2011;69:780–7. Norton WH, Folchert A, Bally-Cuif L. Comparative Analysis of Serotonin Receptor (HTR1A/ HTR1B Families) and Transporter (slc6a4a/b) Gene Expression in the Zebrafish Brain; 2008. Nunes-de-Souza V, Nunes-de-Souza RL, Rodgers RJ, Canto-de-Souza A. 5-HT2 receptor activation in the midbrain periaqueductal grey (PAG) reduces anxiety-like behaviour in mice. Behav Brain Res 2008;187:72–9. Palminteri S, Clair A, Mallet L, Pessiglione M. Similar improvement of reward and punishment learning by serotonin reuptake inhibitors in obsessive-compulsive disorder. Biol Psychiatry 2012;72:244–50. Panula P, Chen YC, Priyadarshini M, Kudo H, Semenova S, Sundvik M, et al. The comparative neuroanatomy and neurochemistry of zebrafish CNS systems of relevance to human neuropsychiatric diseases. Neurobiol Dis 2010;40:46–57.

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