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Contents lists available at ScienceDirect

Neuropharmacology journal homepage: www.elsevier.com/locate/neuropharm

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Effects of lorazepam and WAY-200070 in larval zebrafish light/dark choice test

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Fengjiao Chen, Sijie Chen, Shanshan Liu, Cuizhen Zhang, Gang Peng* Institutes of Brain Science, State Key Laboratory of Medical Neurobiology and Collaborative Innovation Center for Brain Science, Fudan University, Shanghai 200032, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 3 September 2014 Received in revised form 28 February 2015 Accepted 23 March 2015 Available online xxx

Zebrafish larvae spend more time in brightly illuminated area when placed in a light/dark testing environment. Here we report that the anxiolytic drugs lorazepam and diazepam increased the time larval fish spent in the dark compartment in the light/dark test. Lorazepam did not affect the visual induced optokinetic response of larval fish. Gene expression levels of c-fos and crh were significantly increased in the hypothalamus of fish larvae underwent light/dark choice behavior, whilst lorazepam treatment alleviated the increased c-fos and crh expressions. Furthermore, we found estrogen receptor b gene expression level was increased in fish larvae underwent light/dark choice. We next examined effects of estrogen receptor modulators (estradiol, BPA, PHTPP, and WAY-200070) in the light/dark test. We identified WAY-200070, a highly selective ERb agonist significantly altered the light/dark choice behavior of zebrafish larvae. Further investigation showed WAY-200070 treatment caused a reduction of crh expression level in the hypothalamus, suggesting activation of ERb signaling attenuate the stress response. Interestingly, WAY-200070 treatment caused marked increase of c-fos expression in the habenula of fish larvae underwent behavior test. These results suggest WAY-200070 activation of ERb mediated signaling may regulate anxiety related behavior in zebrafish through modulation of neuronal activity in habenula. © 2015 Published by Elsevier Ltd.

Keywords: Anxiety Benzodiazepines Estrogen receptor beta Habenula Zebrafish

1. Introduction The small vertebrate model zebrafish has been increasingly utilized for studying the molecular and neurochemical mechanisms of brain disorders (Kalueff et al., 2014; Mathur and Guo, 2010). Zebrafish exhibit a variety of behaviors including sensorimotor (Burgess and Granato, 2007; Granato et al., 1996), fear and anxiety (Bass and Gerlai, 2008; Bencan et al., 2009), learning and memory (Valente et al., 2012; Yu et al., 2006), and social preference (Engeszer et al., 2004; Saverino and Gerlai, 2008). Anxiety is a sustained response to non-specific and less predictable threats (Davis et al., 2010). Behavioral and pharmacological studies have indicated zebrafish anxiety-like behaviors are similar to those of mammals. The light/dark box test is a widely used method to measure anxiety-like behavior in rodents. In rodent, the light/dark test creates an ethological conflict between exploration

* Corresponding author. Tel.: þ86 21 5423 7817; fax: þ86 21 5423 7643. E-mail address: [email protected] (G. Peng).

and avoidance to brightly lit open area. Anxiolytic compounds such as diazepam and buspirone can reliably increase exploration ac€t, 2003). tivity of rodent in the light/dark test (Bourin and Hascoe Similar to the rodent test, persistent mild stressors such as light induced aversion to brightly illuminated compartment in adult zebrafish (Maximino et al., 2010). In contrast, zebrafish larvae were reported to avoid dark compartment when challenged in the light/ dark box test (Lau et al., 2011; Steenbergen et al., 2011). Neurochemical mechanisms underlying zebrafish anxietyrelated behaviors have been investigated in a number of studies. It was shown the light/dark test increased extracellular and forebrain serotonin levels (Maximino et al., 2013). In adult zebrafish, the light/dark test stimulated c-fos activation in specific brain regions including the stress center hypothalamus and the zebrafish amygdala homolog dorsal telencephalon (Lau et al., 2011). In addition, general stressors such as harsh handling and osmotic shock significantly increased cortisol levels in zebrafish tissues (Alderman and Bernier, 2009; Alsop and Vijayan, 2008). These studies suggest zebrafish anxiety-related behaviors are likely mediated by similar neurochemical mechanisms conserved in vertebrates. It remains to be determined whether anxiolytic

http://dx.doi.org/10.1016/j.neuropharm.2015.03.022 0028-3908/© 2015 Published by Elsevier Ltd.

Please cite this article in press as: Chen, F., et al., Effects of lorazepam and WAY-200070 in larval zebrafish light/dark choice test, Neuropharmacology (2015), http://dx.doi.org/10.1016/j.neuropharm.2015.03.022

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compounds acted through these anxiety-induced neurochemical changes in zebrafish. The present study investigated the effects of anxiolytic compounds in a zebrafish larval light/dark test paradigm. Effects of two benzodiazepam compounds lorazepam and diazepam on zebrafish larval light/dark test were investigated. Further, c-fos activity mapping and expression of corticotropin releasing hormone (crh) were carried out to demonstrate neurochemical changes arisen from light/dark challenge and lorazepam treatment. Because ERb receptor expression was shown to be up-regulated by the light/dark test, estrogen receptor modulators including estradiol valerate, estradiol benzoate, PHTPP, and WAY-200070 were tested for their effect on zebrafish light/dark behaviors. WAY-200070, a highly specific ERb receptor agonist, was further examined for its effect on c-fos and crh gene expressions. 2. Methods 2.1. Animals Zebrafish embryos (TL strain) were collected from breeding tank. Zebrafish larvae were maintained in clear fish water on a 14 h light/10 h dark daily cycle at 28.5  C. The fish water was reverse osmosis purified water dosed to 500 mF salinity with artificial ocean salt mix and buffered to pH7.2 with NaHCO3. Zebrafish larvae were fed a diet of live rotifers between 4 days post fertilization (dpf) and 8 dpf. The rotifers were in-house cultured with Chlorella supplemented with Vitamin B12. The standard lengths of zebrafish larvae used in behavioral analysis were between 3.9 and 4.7 mm. The Fudan University Institutional Animal Care and Use Committee approved all work with zebrafish animals (20110307-084). 2.2. Drugs and reagents Lorazepam, diazepam, estradiol valerate and estradiol benzoate were obtained from the National Institutes for Food and Drug Control of China (NIFDC). PHTPP and WAY-200070 were obtained from Tocris Bioscience. All drugs were dissolved in dimethyl sulfoxide (DMSO) and stored at 20  C. Zebrafish larvae were exposed to the drugs diluted in fish water by soaking and kept in the same solutions during the behavioral test, which lasted 1 h. The soaking exposure (s.e.) required diffusion and/ or ingestion to take effect. For PHTPP and WAY-200070, zebrafish larvae were exposed to the drugs for 45min prior to the behavioral test. Drugs were initially tested at standard concentration of 5 mM and additional concentrations were further tested when appropriate. The final concentration of DMSO in all drug treatment and vehicle control groups was 0.33% (v/v). 2.3. The light/dark test The light/dark test was carried out in light/dark arenas inside a sound-proof wood box. The wood box measured 50  50  75 cm and its interior surface was painted white. The wood box was illuminated inside with white light and the light intensity was 120 lux when measured at the bottom. Zebrafish larvae were individually sucked up with wide-bore plastic Pasteur pipette and transferred to individual plastic dishes (inner diameter 35 mm). The plastic dishes contained clear fish water with drugs or vehicle at indicated concentration. Each plastic dish was then placed in a light/dark arena inside the wood box. The light/dark arena was divided at the middle and the dark compartment was enclosed inside a 5-side black box made from black acrylic plates. The black acrylic plates blocked visible light but allowed transmission of infrared light for monitoring fish movements. Fish movements were recorded through a CCD camera with infrared filter for 1 h. Images were automatically analyzed for fish positions by ZebraLab software from ViewPoint (Viewpoint, France). The software was set up to simultaneously track 8 fish in 8 individual arenas, 4 of which were for vehicle controls and the other 4 were for drug treatment group. The animals for the controls and the treatment group came from the same broods in each test. Results were reported as averaged per 10min period. 2.4. The optokinetic response test Optokinetic response (OKR) is an eye movement behavior elicited by a slowly moving image across the retina. The stimulation is generated by a drum with blackwhite vertical stripes on its internal wall horizontally rotating around the larvae (Roeser and Baier, 2003). The larvae were treated with drugs or vehicle then transferred to a 3.5 cm-diameter petri dish containing 6% methylcellulose at the center of the drum. Images of the optokinetic responses were captured by a CCD camera and processed by ImageJ to analyze the effects of anxiolytic drugs on visual system. 2.5. In situ hybridization Whole-mount in situ hybridization was performed as described (Zhang et al., 2012). Fixed brain tissues of fish larvae were dissect out of the skulls with fine-tip

tweezers. Dissected brain tissues were digested with proteinase K (1:7000, Roche) for 4 h at room temperature. Digoxigenin-labeled antisense RNA probes were hybridized overnight at 68  C. The brain tissues were extensively washed and the hybridized probes were detected with alkaline phosphatase-conjugated antidigoxigenin antibody Fab fragment (1:7500, Roche) and alkaline phosphatase substrate NBT/BCIP (1:80, Roche). Stained brain tissues were cleared and mounted in 100% glycerol for bright-field microscopy. Images were scored by a second experimenter blind to the experimental conditions. To quantify c-fos expression, the percentages of brain tissues with the marked increases of c-fos mapped activity in the hypothalamus were scored. To quantify crh expression, individual crh-expressing cells were counted. Fluorescent in situ hybridization was performed as described (Talbot et al., 2010). DNP-labeled cfos and digoxigenin-labeled crh antisense RNA probes were detected by Tyramide Signal Amplification kits (Invitrogen). Stained brain tissues were mounted in 1% agarose for confocal microscopy. The numbers of animals used for in-situ hybridization were indicted on the corresponding figures. The clones used in this study were constructed by RT-PCR based on sequences and primers below. c-fos: GenBank accession number AL929435. c-fos probe forward: 50 -CGACTTTTGACAGGATGATGTTTACC. c-fos probe reverse: 50 -CCATAGCCCTGCAATGCCATTCG. crh: GenBank accession number BC085458. crh probe forward: 50 -TAACTTGATAGGCAGCAACTAGAAG. crh probe reverse: 50 -GTTACAATGAGACTCAAGTCTGTCAG. 2.6. Western blot Zebrafish larvae post behavior tests were quickly transferred to ice-cold PBS and brain tissues were dissect out of the skulls with fine-tip tweezers. Brain tissues from 24 larvae were lysed and boiled in 1 SDS sample buffer. Denatured and reduced protein samples were resolved on SDS-PAGE gels and then transferred to nitrocellulose membranes. Rabbit anti-CRH (ProteinTech, cat#10944-1-AP) and mouse antib-tubulin (Ab-Mart) antibodies were used at 1:100 and 1:3000 dilutions, respectively. Proteins were detected using a chemiluminescence method (Cell Signaling). Images were acquired with a FluorChem E scanner (Protein Simple). Quantifications of specific protein bands were carried out using Analyze Gel tools in ImageJ. 2.7. Quantitative real-time PCR Zebrafish larvae post behavior tests were stored in a RNA protection reagent until RNA extraction (RNA-later, Sigma). Total RNA was extracted and purified from 16 of 8 dpf zebrafish larvae with the Total-RNA Extract Kit (Sangon Biotech, Shanghai). Extracted RNA was treated by Dnase I (Sigma) to remove residue genomic DNA. cDNAs were obtained using RevertAid H Minus First Strand cDNA Synthesis Kit (Fermentas). Results were normalized to GAPDH and calculated by the normalized CT values (2DDCt). Seven or eight groups of animals were analysed. The primer sequences used for RT-PCR were: GAPDH (control) Forward primer: 50 -CATCGTTGAAGGTCTTATGAGCACTGT. Reverse primer: 50 -AGGTTTCTCAAGACGGACTGTCAG. c-fos: Forward primer: 50 - AGCAGACGAGCAAGGAAATACAAGAC. Reverse primer: 50 - ATGAAGAGATCGCCGTGACAGTTG. esr2b: Forward primer: 50 - AGACTGGAAAACTGATGGATGGATG-30 Reverse primer: 50 - TGCTGTACGATCTTCTGCTAGAGATG-30 2.8. Statistical analyses The statistical analyses were carried out with SPSS20. Data were presented as means ± standard errors. Differences were tested for significance by one-way ANOVA, followed by a post hoc test with Bonferroni correction when appropriate.

3. Results 3.1. Effects of lorazepam on light/dark choice in larval zebrafish Zebrafish larvae spent less time in the dark area when challenged with light/dark environment. Compared to vehicle-treated controls, administration of the GABAA receptor allosteric agonist lorazepam significantly increased the entries and time spent in the dark compartment. These effects were observed in larval fish soaked in water containing 2 mM of lorazepam (entries: F1,30 ¼ 5.167, P ¼ 0.030 and duration: F1,30 ¼ 4.993, P ¼ 0.033). Increasing lorazepam to 5 mM further increased the entries and the time spent in the dark compartment (entries: F1,30 ¼ 17.61, P < 0.001 and time spent: F1,30 ¼ 24.38, P < 0.001). Administration of 10 mM lorazepam caused similar increases in the entries and the

Please cite this article in press as: Chen, F., et al., Effects of lorazepam and WAY-200070 in larval zebrafish light/dark choice test, Neuropharmacology (2015), http://dx.doi.org/10.1016/j.neuropharm.2015.03.022

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time spent in the dark compartment as did the 5 mM lorazepam treatment (entries: F1,30 ¼ 15.90, P < 0.001 and time spent: F1,30 ¼ 15.22, P < 0.001). In contrast to increased entries and time spent in the dark compartment, total locomotions of larval fish in the light/dark box were not significantly affected by these lorazepam treatments (P > 0.05). Lorazepam (5 mM, s.e.) had no significant effects on visual induced behaviors in the optokinetic response test. The saccade rate, eye tracking amplitude and velocity in response to moving white/black bars were not significantly different between the vehicle treated controls and the lorazepam treatment group (Fig. 1d, P > 0.05). 3.2. Effects of lorazepam on c-fos and crh expression In situ hybridization studies of tissue from control animals placed in an evenly lighted-box showed the presence of low to moderate levels of c-fos mRNA in the forebrain, and the presence of low levels of c-fos mRNA in the hypothalamic and hindbrain regions. Compared to these controls, zebrafish challenged with light/ dark choice box showed increases in c-fos mRNA in hypothalamus and hindbrain, with no apparent effect in forebrain. Lorazepam treatment (5 mM, s.e.) abolished the increases of c-fos mRNA in the hypothalamus of zebrafish underwent light/dark test (Fig. 2a). To quantify these effects, the percentages of brain tissues with the marked increases of c-fos mapped activity in the hypothalamus from 7 sets of experiments were analyzed by ANOVA (Fig. 2b). Significant differences were observed (F2,18 ¼ 12.93, P < 0.001). Post hoc tests with Bonferroni corrections showed light/dark test increased hypothalamic activity comparing to controls (P < 0.001). Lorazepam treatment significantly decreased hypothalamic activity (P ¼ 0.005), and restored the c-fos mapped activity to the levels seen in the controls (P ¼ 0.701). Light/dark test and lorazepam treatment had similar effects on expression levels of corticotropin releasing hormone (crh) (Fig. 2c).

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The numbers of crh-positive cells in the hypothalamic region were significantly different among treatment groups (Fig. 2d, F3,139 ¼ 44.47, P < 0.001). Post hoc tests with Bonferroni corrections showed crh expression levels were increased in the hypothalamus of larval fish underwent light/dark test (P < 0.001). Administration of 5 mM lorazepam caused a reduction in crh expression levels in the hypothalamus compared to vehicle treated group (P < 0.001), but the reduced crh expression levels were still significantly higher than controls placed in evenly lighted box (P < 0.001). Increasing lorazepam concentration to 10 mM caused a small further reduction in crh expression levels. Western blotting analysis showed CRH protein levels were similarly affected by these treatments (Fig. 2e). In addition, double fluorescent in-situ hybridization results showed co-expression of c-fos and crh in cells in the hypothalamic region in animals underwent the light/dark test (Fig. 2f). 3.3. c-fos and esr2b expression: qPCR Gene expression profiling suggested ERb receptor gene esr2b expression level was increased in zebrafish challenged with light/ dark test. In qPCR experiments (Fig. 3), compared to controls in evenly illuminated all light boxes, zebrafish in light/dark box tests showed an increase in esr2b mRNA (F1,14 ¼ 5.505, P ¼ 0.034). In agreement with the in situ hybridization data, c-fos mRNA was significantly elevated in zebrafish underwent light/dark test (F1,12 ¼ 9.401, P ¼ 0.010). 3.4. Effects of estradiols, BPA, PHTPP, and WAY-200070 on light/ dark choice To investigate the role of ERb receptor in the light/dark test, animals were treated with estrogen receptor modulators (Fig. 4). Synthetic estrogen analog estradiol valerate somewhat increased the dark entries and the dark duration but the effects were not significant (P > 0.05). Similarly, estradiol benzoate and bisphenol A

Fig. 1. Effects of lorazepam on (A) entries on the dark compartment, (B) time spent in the dark compartment, (C) total distance travelled in the light/dark test, and on (D) visual stimulus induced saccade responses in the optokinetic response test. s.e.: soaking exposure. Rel. Perform.: relative performance normalized to mean values in vehicle controls. Data are mean ± standard error. *, P < 0.05 vs. vehicle control; **, P < 0.01 vs. vehicle control.

Please cite this article in press as: Chen, F., et al., Effects of lorazepam and WAY-200070 in larval zebrafish light/dark choice test, Neuropharmacology (2015), http://dx.doi.org/10.1016/j.neuropharm.2015.03.022

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Fig. 2. Effects of lorazepam on hypothalamic c-fos (A, B) and crh (CeF) expression. Lorazepam was used at 5 mM (A, B, C, D) or 10 mM (D, E, F), soaking exposure. Sagittal views of the whole brain tissue are presented on the left panels. Ventral views of the hypothalamic regions are presented on the right panels (A, C). Dashed circles indicate hypothalamic regions in the sagittal and the ventral views. Data in (B, D) are mean ± standard error. The number of animals used under each condition is indicated at the bottom of corresponding bar. **, P < 0.01 vs. vehicle controls in evenly illuminated all light boxes. ##, P < 0.01 vs. vehicle controls in light/dark tests. Quantification of protein brands in (E) is plotted below the blot image. The CRH/tubulin values were normalized to that of vehicle (Veh.) treated group under all light condition. Figure panels in (F) are maximum intensity projections of confocal image stacks. Figure inserts in the middle panel shows enlarged view of cells from single confocal planes. Scale bar: 100 mm. For figure inserts in (F), scale bar: 10 mm.

(BPA) treatment had no significant effects on the light/dark choice behavior (P > 0.05). Selective estrogen ERb receptor antagonist PHTPP decreased the entries and time spent in the aversive dark compartment but these decreases were not statistically significant (P > 0.05). Administration of PHTPP caused a significant reduction in the total locomotion (F1,22 ¼ 29.66, P < 0.001). The selective ERb receptor agonist WAY-200070 increased the entries and time spent in the dark compartment (Fig. 5). These

increases by WAY-200070 were dose-dependent. Larval fish soaked in water containing 2 mM of WAY-200070 exhibited increased entries and time spent in dark compartment but the increases were not statistically significant (P > 0.05). Increasing WAY-200070 to 5 mM caused further and significant increases in the entries (F1,30 ¼ 37.70, P < 0.001) and the time spent in dark compartment (F1,30 ¼ 25.43, P < 0.001). Effects of WAY-200070 on the entries and the time spent in the dark seemed plateaued at 10 mM (P > 0.05

Please cite this article in press as: Chen, F., et al., Effects of lorazepam and WAY-200070 in larval zebrafish light/dark choice test, Neuropharmacology (2015), http://dx.doi.org/10.1016/j.neuropharm.2015.03.022

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small increase of c-fos mRNA in the habenula compared to even illumination but the increase was not statistically significant (P > 0.05, post hoc with Bonferroni correction). WAY-200070 treatment significantly decreased crh expression in the hypothalamus of fish underwent light/dark test (Fig. 6d, P < 0.001, post hoc with Bonferroni correction, F2,107 ¼ 87.55). The reduced crh expression levels were still significantly higher than controls placed in evenly illuminated box (P < 0.001, post hoc with Bonferroni correction).

4. Discussion

Fig. 3. Effects of light/dark tests on c-fos and esr2b mRNA abundance. Data are mean ± standard error. *, P < 0.05 vs. all light control. The number of animals used is indicated at the bottom of corresponding bar.

comparing to larvae treated with 5 mM of WAY-200070). In these tests, total locomotion was not affected by WAY-200070 treatments (P > 0.05). 3.5. Effects of WAY-200070 on c-fos and crh expression: in situ hybridization Compared to vehicle-treated animals, administration of WAY200070 did not significantly change c-fos levels in the hypothalamus of fish underwent light/dark test (Fig. 6A and B, P > 0.05, post hoc with Bonferroni correction, F2,11 ¼ 12.89). WAY-200070 caused marked increase of c-fos mRNA in the habenula in animals underwent light/dark test (Fig. 6A and B, P < 0.001, post hoc with Bonferroni correction, F2,11 ¼ 142.1). Light/dark test tended to cause a

The present study demonstrated that benzodiazepines alleviated zebrafish larvae's aversion to dark compartment in the light/ dark environment. The behavioral changes by benzodiazepines were not due to general motor or sensory impairment, because lorazepam did not affect fish larval locomotion or visual discrimination dependent optokinetic response. The aversion behavior to dark environment in zebrafish larvae was different from those of adult zebrafish which showed aversion to light compartment in the light/dark test (Lau et al., 2011; Maximino et al., 2010; Steenbergen et al., 2011). The neural mechanisms underlying this developmentally related choice reversal were currently not known (Lau et al., 2011). In mammals, GABAA receptor activation exhibits functionreversal from excitatory in immature neurons to inhibitory in mature neurons (E-I switch). In addition, subunit composition of GABAA receptor has age- and region-specific changes during development that may influence age-adapted function in the brain (Ben-Ari et al., 2007; Galanopoulou, 2008). A recent study showed in zebrafish retinal ganglion cells the E-I switch of GABAergic action occurred during 2e4 dpf (Zhang et al., 2010), a time much earlier than the time period of behavioral choice reversal (between 2 wpf

Fig. 4. Effects of estrogen receptor modulators on (A) entries on the dark compartment, (B) time spent in the dark compartment, (C) total distance travelled in the light/dark test. Data are mean ± standard error. **, P < 0.01 vs. vehicle control.

Fig. 5. Effects of WAY-200070 on (A) entries on the dark compartment, (B) time spent in the dark compartment, (C) total distance travelled in the light/dark test. Data are mean ± standard error. *, P < 0.05 vs. vehicle control; **, P < 0.01 vs. vehicle control.

Please cite this article in press as: Chen, F., et al., Effects of lorazepam and WAY-200070 in larval zebrafish light/dark choice test, Neuropharmacology (2015), http://dx.doi.org/10.1016/j.neuropharm.2015.03.022

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Fig. 6. Effects of WAY-200070 treatment on c-fos (A, B) and crh (C, D) expression. WAY-200070 was used at 5 mM by soaking exposure. Sagittal views of the whole brain tissue are presented on the left panels (A, C). Dorsal view of the habenula regions (dashed circle) are presented on the right panels in (A). Ventral views of the hypothalamic regions are presented on the right panels in (C). Dashed circles indicate hypothalamic regions in the sagittal and the ventral views. Data in (B, D) are mean ± standard error. The number of animals used under each condition was indicated at the bottom of corresponding bar. **, P < 0.01 vs. vehicle controls in evenly illuminated all light boxes. ##, P < 0.01 vs. vehicle controls in light/dark tests. Scale bar: 100 mm.

and 1 mpf) in the light/dark test (Lau et al., 2011). It is not known when the E-I switch takes place in other brain regions in zebrafish. Further studies will be required to determine if the GABA E-I switch plays a role in the developmental related choice reversal in the zebrafish light/dark test. Benzodiazepines can reduce the aversion behavior in both fish larvae and adult, suggesting the larval aversion behaviors are also anxiety-related. Anxiety-related behaviors such as thigmotaxis and its pharmacological modulations have been previously demonstrated in zebrafish larvae (Richendrfer et al., 2012; Steenbergen et al., 2011). The current study further showed by c-fos activity mapping and crh gene expression that compared to evenly illuminated environment, light/dark challenge increased neural activity and crh mRNA in the hypothalamus. It was known c-fos marked neural activities and crh gene expression were elevated in the hypothalamus regions in rodents subjected to acute stress or anxiety-related tests (Duncan et al., 1996; Imaki et al., 1995b). Previous studies also showed chlordiazepoxide reduced stress or novelty induced c-fos activity in most brain areas in rodents (Bechtholt et al., 2008; Imaki et al., 1995a). In the fish larvae light/ dark test, lorazepam treatment reduced c-fos marked neural activities in broad brain regions. Thus the ligh/dark environment likely exerted a sustained mild stress to induce anxiety-related

state in fish larvae and the benzodiazepines can attenuate the anxious state by reducing the neural activity in broad regions in the brain. Quantitative PCR studies demonstrated estrogen receptor b receptor gene esr2 expression level was increased in zebrafish challenged with light/dark test. Estrogen has pronounced effects on multiple mood related behaviors such as depression and anxiety in both females and males (Walf and Frye, 2006; Weiser et al., 2008). Estrogen can exert anxiolytic and anxiogenic actions. Its effects are mainly mediated by two receptors, ERa and ERb. Previous studies have shown that the anxiolytic action of estrogen is likely mediated by ERb. Increased anxiety was observed in ERb-deficient female mice (Krezel et al., 2001). ERb specific agonists such as DPN and WAY-200070 have anxiolytic activities in multiple anxiety related tasks in both male and female mice (Hughes et al., 2008; Lund et al., 2005). ERb is highly expressed in the paraventricular nucleus in hyptothalamus (Laflamme et al., 1998; Shughrue et al., 1997) and ERb agonists can reduce stress-induced increases in corticosterone and ACTH levels in mice (Lund et al., 2006; Oyola et al., 2012; Weiser et al., 2009). Similar to these phenomena in rodents, administration of WAY-207000 in zebrafish light/dark test reduced anxiety related behavior and caused reduction of crh expression in the hypothalamus. Interestingly, circulating corticosterone can up-

Please cite this article in press as: Chen, F., et al., Effects of lorazepam and WAY-200070 in larval zebrafish light/dark choice test, Neuropharmacology (2015), http://dx.doi.org/10.1016/j.neuropharm.2015.03.022

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regulate ERb expression level in rats (Isgor et al., 2003). Thus ERb activation may modulate the neuroendocrine response in HPA axis and the up-regulation of ERb expression under stress may serve to control brain's reaction to obnoxious stimuli. WAY-207000 in zebrafish light/dark test reduced c-fos marked neural activity in the hypothalamus. In contrast to very broad reduction of c-fos activation by lorazepam treatment, WAY-207000 did not have such broad effect. Consistent with findings presented here, previous studies in rodent models showed that ERb agonist treatments (S-DPN or WAY-207000) can reduce c-fos expression in the paraventricular nucleus of hypothalamus (Weiser et al., 2009). WAY-207000 also increases the c-fos expression in the striatum tissue (Hughes et al., 2008), while S-DPN increases c-fos expression in other brain regions involved in the modulation of anxiety-like behavior such as anterodorsal medial amygdala and bed nucleus of the stria terminalis in mice (Oyola et al., 2012). It has been postulated that increased c-fos expression upon ERb activation is likely required for the agonists' effect on anxiety, since a 1.5 h pretreatment is needed to show the effect (Hughes et al., 2008). Indeed, in the zebrafish light/dark test, a pretreatment of 45min was required to demonstrate its positive effect on the anxietyrelated behavior. Thus, unlike the broad dampening effects of benzodiazepine on the neural activity, ERb activation likely involved reduction of activity in the hypothalamus and concurrent elevations of activities in other specific brain regions. c-fos activity mapping also demonstrated a marked increase in the neural activity in the habenula in zebrafish challenged with light/dark test. Habenula is an ancient dorsal thalamic structure conserved in vertebrates (Sutherland, 1982). Neurons in habenula are tuned to visual, olfactory, and nociceptive pain inputs (Dreosti et al., 2014; Gao et al., 1996; Jetti et al., 2014). Importantly, habenula is a hub connecting the forebrain limbic system to the brain stem monoaminergic neurons (Hikosaka, 2010). Activation of habenula may exert a rapid and strong inhibition on the monoaminergic neurons in the brain stem (Christoph et al., 1986; Matsumoto and Hikosaka, 2007; Wang and Aghajanian, 1977). Multiple studies have demonstrated that habenula plays important roles in broad ranges of mood and emotion related behaviors such as aversion, reward, depression, and anxiety through its regulation of the monoaminergic neurons. For example, early habenula lesion studies showed rodents with habenula lesions received more shocks in an active avoidance task (Rausch and Long, 1974). In a recent study, it was found in macaque monkey that a presentation of punishment or an absence of reward activated habenula neurons and subsequently inhibited its downstream dopaminergic neurons (Matsumoto and Hikosaka, 2008). However, the neural mechanisms underlying habenula's roles in animal behaviors are not completely understood. For instance, microinjection of glutamate into the lateral habenula reduces pain threshold in rat whilst injection into the medial habenula increases the pain threshold (Zhao and Wang, 1995). In anxiety related behaviors, the involvement of habenula has been noted. Anxiogenic FG-7142 treatment or lightpotentiated startle can increase the c-fos marked activity in the habenula in rats (Kurumaji et al., 2003; Veening et al., 2009). These results were consistent with our result showing the light/dark test tended to cause a small increase of c-fos mRNA in the habenula. Lesion of habenula efferent pathway in rats increased plasma corticosterone levels and heightened anxiety-related behaviors (Murphy et al., 1996). In the mouse model, genetic ablation of habenula also resulted in an increase in anxiety levels (Kobayashi et al., 2013), while genetic ablation of the triangular septum input into habenula may result in a decrease in anxiety levels (Yamaguchi et al., 2013). In zebrafish model, ablation of the synaptic transmission of medial habenula neurons increased baseline anxiety and aggravated anxiety-like response to stressor (Mathuru and

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66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 Uncited reference 84 85 Egan et al., 2009, Levin et al., 2007. Q1 86 87 Acknowledgments 88 89 This work was supported by the National Natural Science 90 Foundation of China (Grant 31171074) and by the Pujiang Talent 91 Project (Grant 09PJ1401900). 92 93 References 94 Alderman, S.L., Bernier, N.J., 2009. Ontogeny of the corticotropin-releasing factor 95 system in zebrafish. Gen. Comp. Endocrinol. 164, 61e69. http://dx.doi.org/ 96 10.1016/j.ygcen.2009.04.007. 97 Alsop, D., Vijayan, M.M., 2008. Development of the corticosteroid stress axis and 98 receptor expression in zebrafish. Am. J. Physiol. Regul. Integr. Comp. Physiol. 294, R711eR719. http://dx.doi.org/10.1152/ajpregu.00671.2007. 99 Bass, S.L.S., Gerlai, R., 2008. Zebrafish (Danio rerio) responds differentially to 100 stimulus fish: the effects of sympatric and allopatric predators and harmless 101 fish. Behav. Brain Res. 186, 107e117. http://dx.doi.org/10.1016/j.bbr.2007.07.037. Bechtholt, A.J., Valentino, R.J., Lucki, I., 2008. Overlapping and distinct brain regions 102 associated with the anxiolytic effects of chlordiazepoxide and chronic fluoxe103 tine. Neuropsychopharmacology 33, 2117e2130. http://dx.doi.org/10.1038/ 104 sj.npp.1301616. Bencan, Z., Sledge, D., Levin, E.D., 2009. Buspirone, chlordiazepoxide and diazepam 105 effects in a zebrafish model of anxiety. Pharmacol. Biochem. Behav. 94, 75e80. 106 http://dx.doi.org/10.1016/j.pbb.2009.07.009. 107 Ben-Ari, Y., Gaiarsa, J.L., Tyzio, R., Khazipov, R., 2007. GABA: a pioneer transmitter that excites immature neurons and generates primitive oscillations. Physiol. 108 Rev. 87, 1215e1284. 109 €t, M., 2003. The mouse light/dark box test. Eur. J. Pharmacol. 463, Bourin, M., Hascoe 110 55e65. Burgess, H.A., Granato, M., 2007. Sensorimotor gating in larval zebrafish. J. Neurosci. 111 27, 4984e4994. http://dx.doi.org/10.1523/JNEUROSCI.0615-07.2007. 112 Christoph, G.R., Leonzio, R.J., Wilcox, K.S., 1986. Stimulation of the lateral habenula 113 inhibits dopamine-containing neurons in the substantia nigra and ventral 114 tegmental area of the rat. J. Neurosci. 6, 613e619. Davis, M., Walker, D.L., Miles, L., Grillon, C., 2010. Phasic vs sustained fear in rats and 115 humans: role of the extended amygdala in fear vs anxiety. Neuro116 psychopharmacology 35, 105e135. http://dx.doi.org/10.1038/npp.2009.109. 117 Dreosti, E., Vendrell Llopis, N., Carl, M., Yaksi, E., Wilson, S.W., 2014. Left-right asymmetry is required for the habenulae to respond to both visual and olfac118 tory stimuli. Curr. Biol. 24, 440e445. http://dx.doi.org/10.1016/ 119 j.cub.2014.01.016. 120 Duncan, G.E., Knapp, D.J., Breese, G.R., 1996. Neuroanatomical characterization of Fos induction in rat behavioral models of anxiety. Brain Res. 713, 79e91. 121 Egan, R.J., Bergner, C.L., Hart, P.C., Cachat, J.M., Canavello, P.R., Elegante, M.F., 122 Elkhayat, S.I., Bartels, B.K., Tien, A.K., Tien, D.H., Mohnot, S., Beeson, E., 123 Glasgow, E., Amri, H., Zukowska, Z., Kalueff, A.V., 2009. Understanding behavioral and physiological phenotypes of stress and anxiety in zebrafish. Behav. 124 Brain Res. 205, 38e44. http://dx.doi.org/10.1016/j.bbr.2009.06.022. 125 Engeszer, R.E., Ryan, M.J., Parichy, D.M., 2004. Learned social preference in zebrafish. 126 Curr. Biol. 14, 881e884. http://dx.doi.org/10.1016/j.cub.2004.04.042. Galanopoulou, A.S., 2008. GABA(A) receptors in normal development and seizures: 127 friends or foes? Curr. Neuropharmacol. 6, 1e20. 128 Gao, D.M., Hoffman, D., Benabid, A.L., 1996. Simultaneous recording of spontaneous 129 activities and nociceptive responses from neurons in the pars compacta of 130 substantia nigra and in the lateral habenula. Eur. J. Neurosci. 8, 1474e1478. Jesuthasan, 2013). Thus the habenula may serve to control animals' reaction to stress and other obnoxious stimuli and activation of habenula may relieve the anxiety-like state in animals. Here we showed that WAY-200070 played an anxiolytic role and specifically activated habenula neuronal activity. Because ERb is expressed in the habenula (Shughrue et al., 1997), WAY-200070 may act directly on the habenula neurons to modulate their activities. Together, these results suggest the WAY-200070 may potentiate specific habenula activity to alleviate anxiety-related behavior. In conclusion, benzodiazepines and ERb agonist WAY-200070 both function as anxiolytic in the larval light/dark test. While benzodiazepines have broad dampening effects on the neural activity, WAY-200070 may act through activation of specific brain regions including habenula. Further studies will be required to determine how WAY-200070 and ERb regulate the habenula activities to exert the anxiolytic effect.

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