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 citalopramexposed 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.

Examining behavioural test sensitivity and locomotor proxies of anxiety-like behaviour in zebrafish

“…These results imply that measurements of locomotion and anxiety-like behaviour variables may not be related and locomotion as a proxy for anxiety-like behaviour requires further investigation. This is not the first study to have found varying sensitivities in behavioural tests in zebrafish 10,29,30 . There are likely many factors that moderate the effects of anxiolytic compounds on behaviour such as the use of habituation tanks, diet, testing environments, fish source, whether the fish were bred in a lab or caught in the wild, or the presence and proximity of experimenters in the testing room, to name a few.…”

“…By contrast, the GABA A receptor agonist, chlordiazepoxide, binds to benzodiazepine binding sites on GABA A receptor complexes in the central nervous system resulting in an increased binding of GABA to the GABA A receptor 42 . Chlordiazepoxide was synthesized in the late 1950s and has been used 43,44 in many organisms such as mice, rats, dogs, monkeys, rabbits 45 , zoo animals 43 , and recently zebrafish 10,22,29,30,46 . In zebrafish, previous studies have found that chlordiazepoxide has sedative effects 29 , reduces locomotor speed 46 , and increases exploratory behaviour 10 .…”

“…In animals, fear or anxiety is typically exhibited as increased muscle tension, avoidance or escape behaviour, and an increase or surge of autonomic activity 9 . To assess the effects of anxiolytic substances on zebrafish behaviour, previous studies have used several behavioural models adapted from validated rodent paradigms as measures of anxiety-like responses 7,8,10 . Popular behavioural tests used to quantify anxiety-like behaviours in zebrafish include the novel tank dive test 6,8,[11][12][13][14] , the light/dark test 15,16 , and group shoaling [17][18][19][20] , which assess inherent variables of interest specific to each test.…”

Is environmental enrichment beneficial for laboratory animals? A systematic review of studies in zebrafish

“…In general, when exposed to the light/dark apparatus, the adult zebrafish presents a preference for the dark compartment, while avoiding the light compartment (Maximino et al, 2011). Interestingly, anxiolytic drugs increase the time that zebrafish spend in the light compartment while anxiogenic drugs decrease this time (Sackerman et al, 2010). Manuel and collaborators (Manuel et al, 2015) found that zebrafish raised in an EE tank since hatching spent more time in the light compartment in the light-dark test when compared with zebrafish raised in a barren tank since hatching, which suggests that EE decreased anxiety-like behavior.…”

Phenotypic architecture of sociality and its associated genetic polymorphisms in zebrafish

“… 25 , 26 In parallel, zebrafish lines (e.g., AB, TU and WIK) have already been shown to vary in many behaviors, some of them interlinked, including locomotor activity, anxiety traits, stress reactivity, learning abilities and shoaling. 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 The paralleled variation in genetic diversity 25 , 26 and several behavioral phenotypes, provides the rationale that constitutive genetic variation may contribute to the observed behavioral variability.…”