Pharmacology, Biochemistry and Behavior 127 (2014) 82–89

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Strain-dependent differential behavioral responses of zebrafish larvae to acute MK-801 treatment Xiuyun Liu a,1, Ning Guo b,⁎,1, Jia Lin a, Yinglan Zhang a, Xiao Qian Chen c, Sheng Li d, Lin He d,⁎, Qiang Li a,⁎ a Translational Medical Center for Development and Disease, Institute of Pediatrics, Shanghai Key Laboratory of Birth Defect, Children's Hospital of Fudan University, 399 Wanyuan Road, Shanghai 201102, China b Center for Chinese Medical Therapy and Systems Biology, Shanghai University of Traditional Chinese Medicine, 1200 Cailun Road, Shanghai 201203, China c Department of Pathophysiology, School of Basic Medicine, Key Laboratory of Neurological Diseases, Ministry of Education, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China d Bio-X Institutes, Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders, Ministry of Education, Shanghai Jiao Tong University, 1954 Huashan Road, Shanghai 200030, China

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Article history: Received 27 May 2014 Received in revised form 23 October 2014 Accepted 1 November 2014 Available online 6 November 2014 Keywords: Zebrafish Larvae MK-801 Behavior Neurotransmitter

a b s t r a c t The zebrafish is a relatively new model organism and has become a valuable tool in genetic, developmental, and pharmacological researches. Zebrafish larvae, compared with adult, are particularly suitable for high-throughput screening of drug effects. AB and TU are well established in-bred zebrafish strains. The behavioral responses to acute MK-801 treatments (0, 5, 20, 100, and 200 μM) under illumination at 50 lx were studied using zebrafish larvae of both AB and TU strains at 7 dpf with ZebraLab software. Two behavioral parameters, traveling distance and activity counts, were analyzed. “Traveling distance” represents locomotor activity, whereas “activity count” is any activity including small, non-ambulatory movements. Zebrafish larvae of TU strain demonstrated inhibitory effects in both behavioral parameters in response to MK-801 treatment. Zebrafish larvae of AB strain showed lack of responses to MK-801 treatments in traveling distance, and showed increases in activity counts. Therefore, zebrafish larvae of AB and TU strains demonstrated opposite responses in activity counts towards MK-801 treatment. Differences in the level of neurotransmitters and their respective metabolites (NE and MHPG, DA and DOPAC, 5-HT and 5-HIAA) between AB and TU strain zebrafish larvae were discovered by HPLC analysis, which was related to the strain-dependent differential behavioral responses to MK-801 treatment. Conclusion: Under the influences of MK-801, in contrast with TU strain, AB strain zebrafish larvae demonstrated activity changes similar to previous studies on rodents. AB strain larvae are better model organisms than TU strain larvae in MK-801 related behavioral studies. © 2014 Elsevier Inc. All rights reserved.

1. Introduction The zebrafish (Danio rerio) has become a valuable tool not only in genetic and developmental studies (Ackermann and Paw, 2003; Udvadia and Linney, 2003), but also in neuropharmacological researches. The nervous system of zebrafish shares, with higher organisms such as human, some key receptors that are potential drug targets (Rico et al., 2011); therefore, neuroactive drugs were able to influence zebrafish behavior in various ways (Barba-Escobedo and Gould, 2012; Braida et al., 2007; Scerbina et al., 2012; Sison and Gerlai, 2011). It has been demonstrated that both cannabinoid receptor agonist WIN 55,212-2 and serotonin 5-HT1a receptor agonist busporone were able to increase social interaction preference on a PETCO-breed short-fin adult zebrafish (Barba-Escobedo and Gould, 2012; Scerbina et al., 2012; Sison and

⁎ Corresponding authors. Tel./fax: +86 21 64931011. E-mail address: [email protected] (Q. Li). 1 Equal contribution.

http://dx.doi.org/10.1016/j.pbb.2014.11.007 0091-3057/© 2014 Elsevier Inc. All rights reserved.

Gerlai, 2011). In a different study using adult zebrafish with unspecified genetic background, pretreatment with the κ-opioid antagonist, norbinaltorphimine, and the cannabinoid type 1 antagonist, rimonabant, blocked salvinorin A-induced both stimulating and depressive effects on the locomotor activities, and reversed the reinforcing properties of salvinorin A on the conditioned place preference test (Braida et al., 2007). MK-801 (dizocilpine) is a well studied non-competitive NMDA receptor antagonist. Originally discovered as an anti-convulsant (Wong et al., 1986), MK-801 was later found to induce behavioral symptoms that are related to schizophrenia (Javitt and Zukin, 1991). Therefore, MK-801 has been widely used to model schizophrenia (Javitt and Zukin, 1991; Thornberg and Saklad, 1996) in animals to facilitate the mechanistic studies, as well as drug discovery processes. In rodents, MK-801 has been shown to induce hyper-locomotor activities (Ford et al., 1989), stereotypic behaviors (Hitri et al., 1993), disrupted prepulse inhibition of startle responses (Mansbach and Geyer, 1989), and impairments in cognitive functions (Bischoff and Tiedtke, 1992; Highfield et al., 1996). These behavioral changes are considered as

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behavioral hallmarks of MK-801 induced schizophrenic rodent models (Hoffman, 1992; Javitt and Zukin, 1991). Because of the growing importance of zebrafish in neuropharmacological researches, studies with zebrafish on the psychopharmacological properties of MK-801 have been carried out since year 2004 (Seibt et al., 2010; Sison and Gerlai, 2011; Swain et al., 2004). Adult zebrafish from commercial suppliers with unspecified genetic background demonstrated increased circling behaviors under acute treatment of MK-801 at 2 and 20 μM; MK-801 exposure at 2 μM resulted in decreased swimming activities, whereas MK-801 treatment at higher concentrations (20 and 200 μM) did not yield significant changes in swimming activities (Swain et al., 2004). Treatment with 100 μM MK-801 induced an increase in circling behavior in a short fin (SF) wild type breed of adult zebrafish, without affecting the general locomotor activities as measured by bar crossing (Sison and Gerlai, 2011). Exposure to MK-801 at 20 μM caused an increase in locomotor activities in a zebrafish stock with heterogeneous genetic background (Seibt et al., 2010). Although it is still early to say how much these behavioral changes of zebrafish in response to MK-801 treatment are related to schizophrenia, these previously published studies indicated a possibility of using zebrafish as a model organism to study the psychopharmacological effects of MK-801. Compared with adult zebrafish, larval zebrafish are especially suitable for large scale screening of drug effects on behavior. Zebrafish larvae hatch from their chorion on 2 or 3 days post fertilization (dpf). By 5 dpf, the larval nervous system develops sufficiently enough to allow them to adapt and interact with the environment (Budick and O'Malley, 2000; Kimmel et al., 1995). Thereafter a broad range of behaviors, such as thigmotaxis, food intake, escape, and light preference, could be observed (Colwill and Creton, 2011; Pelkowski et al., 2011; Richendrfer et al., 2011; Steenbergen et al., 2011). Recently, increasing number of studies have shown that the larvae are sensitive to drug treatments and many other stimuli (Chen et al., 2010; Ellis et al., 2012; Lee et al., 2012; Richendrfer et al., 2012; Schnorr et al., 2012; Selderslaghs et al., 2010), which provide valuable information to evaluate the effects of drug treatments or other stimuli on neuropsychiatric system. Zebrafish larvae have also been employed as a model organism to elucidate the behavioral effects of MK-801 and its underlying mechanisms (Chen et al., 2010; Turesson et al., 2006). Zebrafish larvae of AB strain demonstrated increased swimming speed after MK-801 exposure, which is consistent with the observations in mammals (Chen et al., 2010). Genetic background is an important factor that influences normal and drug-induced behaviors of zebrafish. Withdrawal from alcohol after chronic administration resulted in abolished shoaling behavior of AB adult zebrafish, while adult zebrafish of SF strain were not affected (Gerlai et al., 2009). Study with AB and Tübingen (TU) strain zebrafish demonstrated significant strain-dependent social behavior patterns, as measured by the reduction of inter-individual distance among shoal members, as zebrafish mature from 7 to 87 dpf. Further HPLC analysis showed different neurotransmitter levels between the two strains (Mahabir et al., 2013a). Adult zebrafish of Nadia, Gaighatta, Scientific Hatcheries, and TM1 zebrafish strains demonstrated significantly different swimming behaviors as measured by their mean horizontal and vertical swimming positions within the tank, when treated with selenium supplement. Strain-dependent differential expression of selenoprotein was also observed in response to selenium supplementation (Benner et al., 2010). Different strains of zebrafish have also been used in recent studies on the neuro-pharmacological properties of MK-801 (Chen et al., 2010; Cognato Gde et al., 2012; Ng et al., 2012; Roberts et al., 2011; Sison and Gerlai, 2011). However, there have been no direct comparisons between the behavioral responses of different strains of zebrafish to MK-801 treatment. Therefore, to better elucidate the influences of genetic background on MK-801 induced behavioral changes in zebrafish larvae, and to facilitate the characterization of zebrafish larvae as model organisms in

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behavioral research, we conducted this comparative study on the behavioral manifestations between zebrafish larvae of AB and TU stains under the influence of MK-801. Two different but related behavioral parameters were analyzed in the current study, locomotor activity and activity quantization. Locomotor activity refers to the movement from one location to another, hence reflecting the purposely positional change of the studied organism. Activity quantization measures the overall activity level of the subject, which not only includes the positional change, but also counts the movements of body parts without relocation of the body, such as tremors and tail movements. For larval zebrafish, whose swimming ability is not fully developed, activity quantization is a more comprehensive indicator of the mobility. In addition to the behavioral experiments, the levels of norepinephrine (NE), dopamine (DA), serotonin (5-HT) and their respective metabolites were analyzed with high precision liquid chromatography (HPLC), as previous studies have reported interactions between MK-801 and above neurotransmitter systems, and the influences of their interactions on rodent locomotor activities (Bubenikova-Valesova et al., 2010; Dai et al., 1995; Huiliang and Carey, 1995; Mathe et al., 1996; Nilsson et al., 2006). 2. Materials and methods 2.1. Zebrafish husbandry Wild type zebrafish of both AB and TU strains were acquired from National Zebrafish Resource of China, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. They were raised and maintained at 28.5 °C according to standard protocols (Westerfield, 1995). Eggs were obtained from second generation of zebrafish by natural spawning, and were raised in groups of 50, in an incubator at 28.5 °C from birth to 7-day post fertilization (dpf) according to published methods (Kimmel et al., 1995). Fish were kept on a 14-h light:10-h dark cycle (lights on at 08:00, lights off at 22:00). All animal experimental procedures were in compliance with local and international regulations. All protocols were approved by the institutional animal care committee of Children's Hospital of Fudan University. 2.2. Drugs MK-801 (M107, Sigma-Aldrich) was dissolved into 10 mM stock solution with sterilized water, and was frozen at − 80 °C in 1 ml aliquots. MK-801 working solution was freshly diluted from concentrated stock to appropriate concentrations with zebrafish system water before experiments. 2.3. Behavior tests All experiments were performed 2 h after the beginning of the light cycle and 2 h before the beginning of the dark cycle in a room with ambient temperature at 28.5 °C. Zebrafish larvae at7 dpf were used in all experiments. Five different MK-801 concentrations (0, 5, 20, 100, and 200 μM) were tested including the 0 μM control group on both AB and TU strains. All groups with different combinations of strains and doses were equally presented in each 48-well plate to avoid any intertreatment variations due to differences in experiment timing and handling. All animals appeared to be normal both during and after the tests. All animals were transferred to new holding tanks, and no death was discovered several weeks after the tests. Zebrafish larvae were carefully transferred to a 48-well plate with one single larva in each well just before the experiment. Excessive fluid was removed, and 250 μl of fresh system water was loaded into each well immediately. Subsequently 250 μl of MK-801 working solution was quickly added into the wells; therefore, each well contained 500 μl liquid. The final MK-801 concentrations were 0, 5, 20, 100, and 200 μM. The plate was immediately placed into Zebrabox (ViewPoint Life Sciences) equipped with a recorder to record the video of zebrafish

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larvae activities, and the activity levels of the larvae were analyzed for 45 min. The video files were saved on the computer for the analysis of zebrafish larvae locomotor activities afterwards. The quantification of zebrafish larvae activity counts was achieved using the quantization mode of ZebraLab software (ViewPoint Life Sciences). The detection threshold was set at 25, an arbitrary level that allowed the software to accurately detect the movement of the larvae. A lower or higher threshold would either cause false recognition or lose track of the larva respectively. The illumination of the testing chamber was set at 50%, which corresponds to 50 lx. After the experiments, the raw files were exported and converted to csv file for further processing. The csv files contained the quantified numeric activity counts (arbitrary units) of each zebrafish larva in every video frame (25 frames per second). The activity count numbers were then compiled into 3 min time bins by simply adding all the numbers within each time bin to give total activity counts of that specific time window. The quantification of zebrafish larvae locomotor activities was achieved using the tracking mode of ZebraLab software with videos recorded from the activity quantization experiments. The same detection threshold (25) in the quantization experiment was used. ZebraLab tracking parameters were used: detection threshold, 25; bin size, 3 min; experiment duration, 45 min; illumination, 50 lx. 2.4. HPLC analysis High precision liquid chromatography (HPLC) was employed to examine the levels of NE and its metabolite 3-methoxy-4hydroxyphenylene glycol (MHPG), DA and its metabolite 3,4dihydroxyphenylacetic acid (DOPAC), 5-HT and its metabolite 5hydroxyindoleacetic acid (5-HIAA). A separate set of zebrafish larvae at 7 dpf without any drug treatment were used for HPLC analysis. Due to the small size of the larvae, the larvae were decapitated and the entire heads instead of the brains were used for the quantification of neurotransmitters. Before decapitation, the fish were rapidly frozen with liquid nitrogen and thawed on ice. No anesthetics were applied in order to avoid the influences of the anesthetics on the levels of the neurotransmitters. For HPLC analysis, 10 samples were acquired for AB strain, and 13 samples were acquired for TU strain. Each sample contained heads of 40 zebrafish larvae. The samples were suspended in ice cold PBS (20 μl/sample) and were homogenized for 4 times (3 s each time) at the speed of 700 rpm on ice. Then the samples were centrifuged at 9600 ×g for 10 min at 4 °C and 1 μl of the supernatant from each sample was used for protein content quantification. Two microliters of stabilizer (0.2 N perchloric acid) was added into each sample and centrifuged again at 9600 ×g for 10 min at 4 °C. The supernatant was collected and stored at − 80 °C. HPLC analysis was carried out using an Agilent 1200 HPLC system (Agilent, USA) with Antec DECARD SDC electrochemical detection (Antec, Netherlands). The column used was Agilent Eclipse XDB-C18 column (5 μM, 4.6 mm × 150 mm). The composition of the mobile phase was as follows: NaH2PO4 (75 mM), OSA (1.7 mM), EDTA (25 μM), TEA (100 μl/l), 10% methanol, and pH 3.5. The detection sensitivities of the neurotransmitters were: NE 0.3 pg/μl, MHPG 0.5 pg/μl, DA 0.7 pg/μl, DOPAC 0.1 pg/μl, 5-HT 0.5 pg/μl, and 5-HIAA 0.2 pg/μl. The internal standards for the neurotransmitters were purchased from Sigma-Aldrich. The percent recovery for each analyte was above 95%. The levels of neurotransmitters were then normalized to the protein content. 2.5. Data presentation and statistics analysis All data presented were expressed as mean ± SEM. Repeated measures two way ANOVA was performed to determine the effects of time, MK-801 doses, and the interactions between the two factors, with “time” as repeated measures. Tukey's multiple comparison posthoc test was performed to compare the means of the groups treated

with different concentrations of MK-801 within each time bin and strain (Figs. 1 and 3). Repeated measures one way ANOVA followed by Tukey's multiple comparison test was used to determine the statistically significant differences between different experiment segments of the control group within each strain, in order to detect the possible habituations (Figs. 1 and 3). Two way ANOVA was employed to determine the significances of strain main effect on both the locomotor activities and activity counts in response to MK-801 treatment, and post test for linear trend was performed (Figs. 2 and 4). Unpaired t-test with Welch's correction was used to determine the statistically significant differences of the neurotransmitter levels between AB and TU zebrafish larvae (Fig. 5). 3. Results 3.1. Effect of MK-801 on the locomotor activity of zebrafish larvae To study the responses of zebrafish larvae to the treatment of MK-801, locomotor activity of zebrafish larvae was examined. The swimming distances of the zebrafish larvae within the 45 min experiment period were first analyzed with time bins set at 3 min (Fig. 1A and c). The recording and the analysis started right after the addition of MK-801 and the transferring of zebrafish larvae into the new environment. It is noticeable that when locomotor activity is measured, zebrafish larvae of both AB and TU strains demonstrated clear habituation as the total distance travelled of the control groups decreased with time to about 50% of the initial values (Fig. 1A and C, closed circle). When the whole 45 min experiment session was divided into three 15 min segments (Fig. 1B and D) and the subsequent segments were compared with the initial segment of the corresponding strain, statistically significant decreases in the travelling distances were observed in the control groups of both AB and TU strains (AB strain: F(2,34) = 11.04, p b 0.05, Fig. 1B, opened bars; TU strain: F(2,34) = 11.59, p b 0.05, Fig. 1D, opened bars). Tukey's multiple comparison post-hoc tests also discovered significant differences between the first segment and the subsequent segments in the control groups of each strain. 3.1.1. Effect of MK-801 on the locomotor activity of AB strain zebrafish larvae As shown in Fig. 1A, compared with 0 μM control group, low doses of MK-801 treatment at either 5 μM or 20 μM did not affect the total distance travelled by AB strain zebrafish larvae in each 3 min time bin. Higher doses of MK-801 treatment at 100 μM and 200 μM only yield an inhibition in the locomotor activity at the early phase of the experiment session. This observation was better demonstrated when the whole 45 min experiment session was divided into three 15 min segments (Fig. 1B). Repeated measures two way ANOVA detected significant contributions of time (F(2,170) = 13.80, p b 0.05), and the interaction between time and MK-801 doses (F(8,170) = 5.616, p b 0.05) to the locomotor activities of the zebrafish larvae, whereas MK-801 doses were determined as non-significant. However, in the first 15 min segment, with the increases in MK-801 concentration, progressively decreased swimming distances of the larvae were observed, and the 200 μM treated group was significantly different from the control group as determined by Tukey's multiple comparison post-hoc test (Fig. 1B, left panel). 3.1.2. Effect of MK-801 on the locomotor activity of TU strain zebrafish larvae Fig. 1C and d showed the results of the TU strain larvae. MK-801 treatment resulted in inhibitory effects on the locomotor activity of the larvae as measured by the swimming distance. As shown in Fig. 1C, the control group demonstrated a gradual decrease in the swimming distance with the progression of the experimental session. Compared with the control group, the MK-801 treated groups showed similar swimming distances in the very first 3 min time bin right after the introduction of MK-801 into the system, and decreased dramatically

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Fig. 1. Swimming distances of zebrafish larvae under the influence of MK-801. Left panels (A and B) represent data of AB strain, and right panels (C and D) represent data of TU strain. a, c: Swimming distances of zebrafish larvae are combined into 3 min time bins. The horizontal axis denotes the progression of the time of the experiments. The vertical axis denotes the total distance travelled within each 3 min time bin by zebrafish larvae. Data are presented as mean ± SEM, n = 18 animals per group. Only error bars above the data points are visualized. To better demonstrate the differences between differently treated groups, groups of 0 μM (control), 5 μM, and 200 μM are shown in black; while groups of 20 μM and 100 μM are shown in gray. b, d: The whole 45 min experiment session is divided into three 15 min segments, as shown by horizontal axis. The total swimming distances of zebrafish larvae in each 15 min segment are calculated and plotted with bar graph. The vertical axis denotes the total distance travelled within each 15 min segment by zebrafish larvae. Data are presented as mean ± SEM, n = 18 animals per group. *, significantly different from control group of the same segment, p b 0.05 (one way ANOVA followed by Tukey's multiple comparison test). #, significantly different from the first segment of the corresponding strain, p b 0.05 (repeated measures one way ANOVA followed by Tukey's multiple comparison test).

afterwards. Treatment with MK-801 at 5 μM exhibited an intermediate inhibition on the swimming distances. Treatments with MK-801 at 20, 100, and 200 μM showed maximum inhibitions, as the inhibition effects did not increase with the increasing dosages of MK-801. The whole 45 min experiment session was divided into three 15 min segments to better compare the effects of MK-801 treatments on the swimming distances of the larvae (Fig. 1D). Repeated measures two way ANOVA

Fig. 2. Normalized total swimming distances of zebrafish larvae under the influence of MK-801. The horizontal axis denotes the concentration of MK-801 treatment. The vertical axis denotes the normalized total swimming distances within the whole 45 min experiment session. Data are presented as mean ± SEM, n = 18 animals per group. Only error bars above the data points are visualized.

discovered significant influences of time (F(2,170) = 47.66, p b 0.05) and MK-801 doses (F(4,85) = 10.63, p b 0.05), as well as their interactions (F(8,170 = 3.594, p b 0.05) on the locomotor activities of the larvae. Tukey's multiple comparisons tests were performed to detect the significant differences between the control group and the MK-801 treated groups within each experiment segment. In the first 15 min segment, statistically significant differences were discovered between the control group and the groups treated with 20, 100, and 200 μM MK-801 (Fig. 1D, left panel). In the second 15 min segment, compared with control group, all MK-801 treated groups demonstrated significantly decreased swimming distances (Fig. 1D, center panel). In the last 15 min segment, no significant difference was discovered (Fig. 1D, right panel). 3.1.3. Differences of effect of MK-801 on the locomotor activity between AB and TU strain zebrafish larvae In order to better explore the differences in the locomotor activities in response to MK-801 treatments between AB and TU strain zebrafish larvae, the total swimming distances of the zebrafish larvae under the influences of MK-801 were normalized to that of the respective nontreated control group (Fig. 2). Normalization eliminated the differences in locomotor activities resulting from different genetic backgrounds between the strains; therefore, the strain-dependent behavioral responses to MK-801 treatment could be isolated. Two way ANOVA discovered significant influences of genetic background on the locomotor responses to MK-801 treatment (F(1,170) = 34.87, p b 0.05). In addition, TU zebrafish larvae demonstrated a significant linear trend with a slope of −0.1523 in the locomotor activities in response to increases in MK801 concentration (post test for linear trend, p b 0.05). No significant linear trend was discovered for AB strain larvae.

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3.2. Effect of MK-801 on the activity counts of zebrafish larvae The activity counts of zebrafish larvae, which reflected the activity levels of zebrafish larvae, were also studied to provide an additional parameter for the analysis of the effect of MK-801. Activity quantization analysis was performed on the same set of recorded experiment videos as the ones used in analysis of locomotor activities. Fig. 3A and C showed the results of AB and TU strain larvae respectively with 3 min time bin. As shown in Fig. 3A and C, the control groups of both AB and TU strain zebrafish larvae demonstrated clear habituation on activity counts, as the curves of activity counts declined with the progression of the experiment (Fig. 3A and C, closed circle). When the whole 45 min experiment session was divided into three 15 min segments (Fig. 3B and D) and the subsequent segments were compared with the initial segment of the corresponding strain, statistically significant decreases in the activity counts were observed in the control groups of both AB and TU strains (Fig. 3B and D, open bars). 3.2.1. Effect of MK-801 on the activity counts of AB strain zebrafish larvae When AB strain larvae were treated with 5 μM MK-801, an early onset moderate up regulation of the activity counts was observed, and the up regulation gradually decreased to control level (Fig. 3A, open circle). When AB strain larvae were treated with higher doses of MK-801 (20, 100, and 200 μM), the increase in activity counts was only observed in the later phase of the experiment starting at about 15 min into the experiment (Fig. 3A). The largest increase in activity counts was observed with the highest dose of MK-801 tested at 200 μM. The increases in activity counts associated with 20 and 100 μM MK-801 treatments were comparable to the initial phase of 5 μM MK-801 treatment (Fig. 3A). This differential time dependent response to MK-801 treatment at different concentrations is better illustrated when the time bin was set to

15 min (Fig. 3B). Repeated measures two way ANOVA was employed to determine the influences of time and MK-801 doses on the activity counts of the larvae. Although both time and MK-801 doses were determined as non-significant, significant influences of the interaction between the two factors were discovered (F(8,170) = 6.800), which indicated a potentially complicated mode of MK-801 actions. Compared with the non-treated control group, post-hoc tests discovered significant increases in activity counts of the group treated with 200 μM MK801 in both the second and the third 15 min segments (Fig. 3B, center and right panels). Although a clear increase in activity counts of the 5 μM MK-801 treated group in the first 15 min segment was observed, the increase was determined as non-significant (Fig. 3B, left panel). 3.2.2. Effect of MK-801 on the activity counts of TU strain zebrafish larvae Instead of the MK-801 induced increases in activity counts observed with AB strain zebrafish larvae, inhibitions of activity counts of TU strain larvae under the treatment of MK-801 were observed (Fig. 3C), which were similar to the inhibitory effects that MK-801 exerted on locomotor activity of TU strain larvae (Fig. 1C). As shown in Fig. 3C, all groups demonstrated decreases in the activity counts with the progression of the experiment. Compared with the control group, the 5 μM MK-801 treated group displayed no inhibitory effect, whereas the 200 μM treated group showed the strongest inhibitory effect. Groups treated with 20 and 100 μM MK-801 exhibited intermediate inhibitory effects in response to MK-801 treatment. When the whole 45 min experiment session was divided into three 15 min segments, with increases in MK-801 concentrations, progressive decreases in activity counts were observed in the first and the second segment (Fig. 3D, left and center panels). Repeated measures two way ANOVA discovered significant influences of time (F(2,170) = 43.10, p b 0.05) and the interaction between time and MK-801 doses (F(8,170) = 2.004, p b 0.05) on the activity counts

Fig. 3. Activity counts of zebrafish larvae under the influence of MK-801. Left panels (A and B) represent data of AB strain, and right panels (C and D) represent data of TU strain. a, c: Activity counts of zebrafish larvae are combined into 3 min time bins. The horizontal axis denotes the progression of the time of the experiments. The vertical axis denotes the activity counts of zebrafish larvae within each 3 min time bin. Data are presented as mean ± SEM, n = 18 animals per group. Only error bars above the data points are visualized. To better demonstrate the differences between differently treated groups, groups of 0 μM (control), 5 μM, and 200 μM are shown in black; while groups of 20 μM and 100 μM are shown in gray. b, d: The whole 45 min experiment session is divided into three 15 min segments, as shown by horizontal axis. The activity counts of zebrafish larvae in each 15 min segment are calculated and plotted with bar graph. The vertical axis denotes the total activity counts of zebrafish larvae in each 15 min segment. Data are presented as mean ± SEM, n = 18 animals per group. *, significantly different from control group of the same segment, p b 0.05 (one way ANOVA followed by Tukey's multiple comparison test). #, significantly different from the first segment of the corresponding strain, p b 0.05 (repeated measures one way ANOVA followed by Tukey's multiple comparison test).

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of the larvae. Although, the effects of MK-801 doses were determined as non-significant, significant decreases in activity counts were discovered between the MK-801 treated group and the control group in the first and second 15 min segments (Fig. 3D, left and center panels). No significant differences were discovered between the control group and the MK-801 treated groups in the third 15 min segment (Fig. 3D, right panel). 3.2.3. Differences of effect of MK-801 on the activity counts between AB and TU strain zebrafish larvae To better explore the differences in the activity counts in response to MK-801 treatments between AB and TU strain zebrafish larvae, the total activity counts of the zebrafish larvae under the influences of MK-801 were normalized to that of the respective non-treated control group (Fig. 4). Two way ANOVA discovered significant influences of genetic background on the activity counts in response to MK-801 treatment (F(1,170) = 20.01, p b 0.05). TU zebrafish larvae demonstrated a significant linear trend with a slope of −0.1804 in the activity counts in response to increases in MK-801 concentration (post test for linear trend, p b 0.05). No significant linear trend was discovered for AB strain larvae. 3.3. Neurotransmitter levels in AB and TU zebrafish larvae at 7 dpf Previous studies have reported interactions between MK-801 and neurotransmitter systems involving NE, DA and 5-HT etc. To explore the possible neurochemical mechanisms underlying the differential responses to MK-801 treatment between AB and TU strain zebrafish larvae, the levels of NE and its metabolite MHPG, DA and its metabolite DOPAC, 5-HT and its metabolite 5-HIAA were measured (Fig. 5). The neurotransmitter contents were normalized to the protein contents of the same respective sample. The samples were harvested from 7 dpf zebrafish larvae of AB and TU strains without any drug treatment, therefore the differences in the neurotransmitter levels were not due to MK801, but reflected the native differences between the two strains. HPLC analysis revealed significant higher levels of NE, MHPG, DOPAC and 5-HT in TU strain larvae than those of AB strain larvae (Fig. 5). 4. Discussion Behavioral analysis is not only a method to study the normal functions of the nervous system, but also a valuable tool in the investigations of neurological disorders of the studied organism (McAllister and Stein, 2010; Wall and Messier, 2001). Changes in normal behavioral patterns often reflect disturbed neuropsychiatric state. Therefore, behavioral animal studies are carried out to facilitate the identification of molecular

Fig. 4. Normalized total activity counts of zebrafish larvae under the influence of MK-801. The horizontal axis denotes the concentration of MK-801 treatment. The vertical axis denotes the normalized total activity counts within the whole 45 min experiment session. Data are presented as mean ± SEM, n = 18 animals per group. Only error bars above the data points are visualized.

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and cellular mechanisms underlying mental disorders and the screening of drug candidates. In this present study, the behavioral responses of zebrafish larvae to acute MK-801 challenges at different concentrations were evaluated with two different zebrafish strains, AB and TU. The two strains demonstrated differential responses to MK-801 treatment. One interesting observation was made when the activity counts of zebrafish larvae were analyzed, in which zebrafish larvae of AB and TU strains demonstrated opposite responses. When treated with MK-801, AB strain zebrafish larvae showed increases in activity counts (Figs. 3A and B and 4), whereas TU strain zebrafish larvae showed decreases (Figs. 3C and d and 4). Therefore, MK-801 treatment increased activity counts of AB zebrafish larvae, and exerted inhibitory effects on TU zebrafish larvae. Another interesting observation was made with AB zebrafish larvae. The increases in activity counts in MK-801 treated groups did not transfer into increases in locomotor activity, which indicated that these two behavioral parameters are related but not synonymous. In rodents, low doses of MK-801 induced hyper-locomotor activities, while high doses of MK-801 strongly inhibited locomotor activities and stimulate stereotypic behaviors and ataxia (Wu et al., 2005). When tested on adult zebrafish, MK-801 at 20 μM induced hyper-locomotor activities in commercial breed with heterogeneous genetic background (Benner et al., 2010). In a different study using an out-bred short fin zebrafish strain, MK-801 treatment did not affect the general locomotor activities of the adult zebrafish even with the highest concentration tested, which is 100 μM (Sison and Gerlai, 2011). The swimming speed of AB zebrafish larvae at the age of 5 dpf increased significantly after 1 h treatment with 50 μM MK-801 (Chen et al., 2010). Our results with TU zebrafish larvae at 7 dpf unequivocally demonstrated motor inhibitory effects of MK-801 treatment across all the concentrations tested. The locomotion of AB zebrafish larvae, as measured by travelling distance, is not quite sensitive to MK-801 treatments. The activity counts of AB zebrafish larvae increased in response to MK-801 treatment, which is in agreement with the role of MK-801 as a psychostimulant. Larval zebrafish is a unique model system; therefore, discoveries from adult zebrafish and rodents should not be readily adopted in studies with zebrafish larvae. More studies are needed in order to establish a comprehensive behavioral profile of zebrafish larvae. AB and TU are well-established and widely used wild type zebrafish strains. Differences in development, as well as behavioral manifestations, between the two strains were discovered both in natural and challenged conditions. Study with AB and TU strain zebrafish demonstrated significant strain-dependent social behavior patterns, as measured by the reduction of inter-individual distance among shoal members, as zebrafish mature from 7 to 87 dpf (Mahabir et al., 2013a). In a different study, TU zebrafish larvae displayed higher locomotor activities than AB larvae during the day time, whereas similar levels of locomotor activities were displayed by the two strains during the night. Adult TU zebrafish demonstrated less anxiety related behaviors than adult AB zebrafish, as measured by the time spent in the top of a novel tank (Vignet et al., 2013). In addition, embryonic alcohol exposure resulted in dosedependent decreases in the levels of dopamine, DOPAC, serotonin, and 5-HIAA in AB strain zebrafish, whereas the levels of the above neurotransmitters were not affected in TU strain zebrafish. However, the concentration of alcohol inside the egg did not differ between the AB and TU zebrafish strains during the exposure (Mahabir et al., 2013b). The differences in response to MK-801 treatment between AB and TU zebrafish larvae could possibly due to the differences in their genetic background and the resulted differences in the development of nervous system. In Mahabir's study, in addition to the differences in the social behavior patterns between AB and TU zebrafish, HPLC analysis showed differences in the expression levels of multiple neurotransmitters between the two strains, which provided evidence of how important genetic factors are (Mahabir et al., 2013a). Furthermore, straindependent differential expression of neurotransmitters and neurotransmitter receptors is also observed between zebrafish of AB and SF

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Fig. 5. Comparisons of neurotransmitter levels between AB and TU strain zebrafish larvae at 7 dpf. Six neurotransmitters were analyzed. a) NE, a′) MHPG, b) DA, b′) DOPAC, c) 5-HT, d) 5-HIAA. The vertical axis denotes the normalized level of neurotransmitters. Data are presented as mean ± SEM, n = 13 groups for TU strain larvae and n = 10 groups for AB strain larvae. Each group contained 40 zebrafish larvae. *, significant difference, p b 0.05 (unpaired t-test with Welch's correction).

strains (Pan et al., 2012). In this current study, different levels of neurotransmitters were also detected between AB and TU strain zebrafish larvae, and could be the potential causes of the differential responses of the two strain zebrafish larvae to MK-801 treatment. Across all the neurotransmitters tested, TU strain zebrafish larvae displayed higher expression levels of NE, MHPG, DOPAC and 5-HT. Our report for the first time characterized the behavioral response of TU zebrafish larvae to the treatment of MK-801, and discovered unexpected inhibitory effects of MK-801 both on locomotion and activity counts. On the contrary, AB zebrafish larvae demonstrated increased activity counts when treated with MK-801, which is consistent with previous studies with rodents. Therefore, AB strain larvae are better model organisms than TU strain larvae in MK-801 related behavioral studies. These differences in the behavioral responses to MK-801 treatment between AB and TU strain zebrafish larvae may be attributed to the differences in the levels of neurotransmitters in their nervous system. Acknowledgments We thank Prof. Albert Chang-Hai Yu from Beijing University, and Ms. Ming Deng, Dr. Xiaolan Peng and Mr. Zhaofu Chang from Ruijin Hospital for advice and assistance. We gratefully acknowledge the assistance of Children's Hospital of Fudan University. This work was supported by

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Strain-dependent differential behavioral responses of zebrafish larvae to acute MK-801 treatment.

The zebrafish is a relatively new model organism and has become a valuable tool in genetic, developmental, and pharmacological researches. Zebrafish l...
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