Sustained Effects of Developmental Exposure to Ethanol on Zebrafish Anxiety-Like Behaviour

Abstract

In zebrafish developmentally exposed to ambient ethanol (20mM-50mM) 1-9 days post fertilization (dpf), the cortisol response to stress has been shown to be significantly attenuated in larvae, juveniles and 6 month old adults. These data are somewhat at variance with similar studies in mammals, which often show heightened stress responses. To test whether these cortisol data correlate with behavioural changes in treated animals, anxiety-like behaviour of zebrafish larvae (9dpf and 10dpf) and juveniles (23dpf) was tested in locomotor assays designed to this end. In open field tests treated animals were more exploratory, spending significantly less time at the periphery of the arena. Behavioural effects of developmental exposure to ethanol were sustained in 6-month-old adults, as judged by assessment of thigmotaxis, novel tank diving and scototaxis. Like larvae and juveniles, developmentally treated adults were generally more exploratory, and spent less time at the periphery of the arena in thigmotaxis tests, less time at the bottom of the tank in the novel tank diving tests, and less time in the dark area in scototaxis tests. The conclusion that ethanol-exposed animals showed less anxiety-like behaviour was validated by comparison with the effects of diazepam treatment, which in thigmotaxis and novel tank diving tests had similar effects to ethanol pretreatment. There is thus a possible link between the hypophyseal-pituitary-interrenal axis and the behavioural actions of developmental ethanol exposure. The mechanisms require further elucidation.

Fig 1

Stress-related behaviour assessed by thigmotaxis in zebrafish larvae A,B) 9dpf, C,D) 10dpf, E,F) 23dpf juveniles. G,H) Effect of diazepam on larval stress-reactivity assessed by thigmotaxis. Time course of average time spent each minute at the edge of the apparatus (A, C, E), overall average time spent per minute at the edge of the apparatus (B, D, F). (A-D) Developmental ethanol exposure decreased thigmotaxis at both 9dpf (A,B: F 2,105 = 4.76, P<0.05) and 10dpf (C,D: F 2, 285 = 6.69, P<0.05), with the greatest difference between 20mM ethanol treatment and the control. Siblings of the same animals were raised for another 2 weeks and tested as 23 dpf juveniles (E,F). These juveniles exhibited a similar thigmotaxis response as at 9dpf, with decreased thigmotaxis in ethanol treated animals compared to controls (F 2,146 = 2.93, P<0.05). (G-H) Larvae acutely treated with diazepam for 6 minutes exhibited significantly reduced time spent at the edges of the wells compared to controls (F 1, 259 = 5.47, P<0.01). There were no significant differences in distance travelled. Post-hoc t-test: *** P<0.001, ** P<0.01.

Fig 2. Stress-reactivity of 6-month old zebrafish as assessed by thigmotaxis.

A) Time course of average time spent per minute at the edge of the apparatus, B) overall average time spent per minute at the edge of the apparatus, C) distance travelled during thigmotaxis, D) Effect of diazepam on adult zebrafish stress-reactivity assessed by thigmotaxis. A,B) Adult zebrafish that had been experimentally exposed to ethanol spent decreased time at the edge of the tank, (F 2, 127 = 3.09, P<0.05). There were no significant differences in distance travelled (C). Adults acutely exposed to diazepam exhibited reduced time spent at the edges of the tanks compared to controls (F 1, 4.98 = 5.44, P<0.001) (D). Post-hoc t-test, * P<0.05.

Fig 3. Stress-reactivity measured by novel tank diving in 6-month old adult zebrafish.

A) Time course of average time spent per minute at the bottom of the tank, B) overall average time spent per minute at the bottom of the tank each minute, C) mean distance travelled per minute during novel tank diving. D) Effect of diazepam on zebrafish stress-reactivity assessed by novel tank diving. A,B. Zebrafish that had been developmentally exposed to ethanol showed reduced bottom dwelling (F 2, 682 = 3.47, P<0.05). There were no significant differences in distance travelled (C). D. Diazepam also significantly reduced time spent by adults at the bottom of the tanks compared to controls (F 1, 408 = 5.45, P<0.001). Post hoc t-test, *** P<0.001.

Fig 4. Stress-reactivity measured by scototaxis in 6-month old adult zebrafish A) Time course of average time spent at the bright side of the apparatus and B) overall average time spent at the bright side of the apparatus.

Adult zebrafish that had been developmentally exposed to ethanol spent more time on the bright side of the tank (F 2, 31 = 3.85, P<0.05). Post hoc t-test, ** P<0.01, * P<0.05.