Zebrafish Behavior in Novel Environments: Effects of Acute Exposure to Anxiolytic Compounds and Choice of Danio rerio Line

Abstract

Zebrafish (Danio rerio) associative responses are useful for pharmaceutical and toxicology screening, behavioral genetics, and discovering neural mechanisms involved in behavioral modulation. In novel environments, zebrafish swim to tank bottoms and dark backgrounds, behaviors attributed to anxiety associated with threat of predation. To examine possible genetic effects of inbreeding and segregation on this behavior, we compared Zebrafish International Resource Center (ZIRC) AB and WIK lines to zebrafish and GloFish® from a pet store (PETCO) in two qualitatively different novel environments: the dive tank and aquatic light/dark plus maze. Behavior was observed in the dive tank for 5 min, immediately followed by 5 min in the light/dark plus maze. Among strains, WIK spent more time in the dive tank top than AB (76 ± 30 vs. 17 ± 11 sec), and AB froze in the plus maze center for longer than PETCO or GloFish® (162 ± 61 vs. 72 ± 29 or 27 ± 27 sec). Further, behavior of zebrafish exposed for 3 min to 25 mg/L nicotine, desipramine, chlordiazepoxide, yohimbine, 100 mg/L citalopram, 0.05% DMSO, or 0.5% ethanol was compared to controls. Approximately 0.1% of drug is available in brain after such exposures. Desipramine or citalopram-exposed fish spent more time in the dive tank top, and both reuptake inhibitors bound to serotonin transporters in zebrafish brain with high affinity (K(i) = 7 ± 5 and 9 ± 5 nM). In the plus maze, chlordiazepoxide, ethanol and DMSO-exposed fish crossed more lines and spent more time in white arms. Neither 25 mg/L nicotine nor yohimbine altered zebrafish behavior in novel environments, but nicotine was anxiolytic at higher doses. Overall, the light/dark plus maze and dive tank are distinct behavioral measures that are sensitive to treatment with anxiolytic compounds, but zebrafish line selection and solvents can influence baseline behavior in these tests.

Figure 1

The novel dive tank. Upon introduction to the dive tank, untreated controls initially dwell in the bottom third of the tank before venturing up to the top 2/3 of the tank (per Levin et al., 2007). Observations are made over 5 min.

Figure 2

The novel aquatic light/dark plus maze. Untreated fish tend to freeze in the center and initially enter black arms when first introduced into the plus maze. After several minutes they begin to explore the maze. Observations are made over 5 min.

Figure 3

Behavior of zebrafish lines and lack of nicotine effect at 25mg/L in dive tank. WIK line zebrafish spent more time in the top 2/3 of the novel dive tank than AB fish, and nicotine treatment had no significant effect. Mean ± S.E.M. are shown. Sample sizes (N) for AB and WIK = 6, GloFish® = 5, and PETCO zebrafish = 8, both for untreated and nicotine treated fish. An * indicates significantly more time in the top of the tank than the AB strain (p < 0.05).

Figure 4

Drug and solvent effects on dive tank exploration. Zebrafish treated for 3 min with 25mg/L desipramine (DMI) spent significantly more time in the top 2/3 of the dive tank than controls (CTRL). Mean ± S.E.M. are shown, N = 8. Fish were obtained from PETCO, Paramus, NJ. An * indicates significantly more time in top 2/3 of tank than controls (p < 0.05). There was a non-significant trend for zebrafish exposed to 0.5% ethanol (EtOH) to spend more time in the top 2/3 of the tank (p = 0.08), indicated by τ Neither 25 mg/L nicotine (NIC) nor 0.05% DMSO affected behavior in the dive tank.

Figure 5

Anxiolytic, but not anxiogenic drug effects on zebrafish behavior in the dive tank. Citalopram exposure for 3 min at 100mg/L resulted in zebrafish spending significantly more time than controls in the top 2/3 of the dive tank. Mean ± S.E.M. are shown, sample size = 9. Fish were obtained from Aquatic-Ecosystems. An * indicates significantly more time in top of tank than controls or other treatment groups (p < 0.001). Exposure to 25 mg/L yohimbine (YOH) or chlordiazepoxide (CDE) did not affect zebrafish vertical localization within the dive tank.

Figure 6

AB line zebrafish exhibited significantly greater latency to enter an arm than PETCO or GloFish zebrafish, but not WIK line zebrafish. Nicotine exposure had no influence on this parameter. Mean + S.E.M. are shown. Sample sizes for AB, WIK and GloFish® = 6, and PETCO zebrafish = 8. An * indicates significantly more time initially frozen in the middle of the plus-maze than GloFish or PETCO zebrafish (ANOVA and Tukey’s HSD post-hoc, p < 0.05).

Figure 7

(a) Zebrafish from PETCO exposed to ethanol (0.5%) or DMSO (0.05%) entered significantly more arms in the aquatic light/dark plus-maze than controls (ANOVA and Fisher’s LSD post-hoc, p < 0.05). Mean ± S.E.M. are shown, sample size = 8, except N = 7 for the EtOH group in which one fish was injured on transfer. Nicotine (NIC) and desipramine (DMI) treatment had no effect on this measure. (b) White time: percent time spent in white arms. PETCO zebrafish exposed to 0.5% ethanol (EtOH) or 0.05% DMSO spent significantly more time in white arms than untreated controls (ANOVA and Fisher’s LSD post-hoc, p < 0.05).

Figure 8

Effects of drugs targeting excitatory and inhibitory neurotransmitter systems on behavior of zebrafish from Aquatic Eco-Systems in the light/dark plus-maze. Mean + S.E.M. are shown, sample size = 9, CTRL = control, YOH = yohimbine 25 mg/L, CDE = chlordiazepoxide 25 mg/L; CIT: citalopram 100 mg/L. Total arm entries did not differ significantly among drug treatment groups, yet (a) chlordiazepoxide-treated zebrafish had proportionally more white arm entries, and (b) spent more time in white arms than untreated controls (ANOVA and Fisher’s LSD post-hoc, p < 0.05).

Figure 9

(a) Saturation binding of [³H] citalopram to serotonin transporters (SERTs) in zebrafish whole brain membranes by non-linear regression (N = 3 assays). Membranes pooled from 10–12 adult mixed-sex zebrafish were incubated with concentrations of [³H] citalopram ranging from 0.1 to 10 nM. Non-specific binding was defined by 20 µM fluoxetine. The K_D of zebrafish brain SERT binding sites is 15.6 ± 5 nM and Bmax is 278 ± 70 fmol/mg protein. (b) Displacement of 2.5 nM [³H] citalopram from zebrafish whole brain membranes by sertraline, desipramine and GBR 12909 (N = 3 assays). Sertraline (Ki = 9 ± 5 nM) and desipramine (Ki = 7 ± 5 nM) exhibit similar high affinities for zebrafish SERT binding sites, while GBR 12909 (Ki > 1000 nM) exhibits negligible affinity for them.