Inhibition of ecto-5'-nucleotidase and adenosine deaminase is able to reverse long-term behavioural effects of early ethanol exposure in zebrafish (Danio rerio)

Abstract

The behavioural impacts of prenatal exposure to ethanol include a lower IQ, learning problems, anxiety and conduct disorders. Several components of the neurochemical network could contribute to the long-lasting effects of ethanol embryonic exposure. Adenosine is an important neuromodulator, that has been indicated to be affected by acute and chronic exposure to ethanol. Here, embryos of zebrafish exposed to 1% ethanol during the developmental stages of gastrula/segmentation or pharyngula exhibited anxiolytic effect, increased aggressiveness, and decreased social interaction. The exposure during pharyngula stage was able to affect all behavioural parameters analysed at 3 months-post fertilization (mpf), while the treatment during gastrula stage affected the anxiety and social interaction parameters. The aggressiveness was the only behavioural effect of early ethanol exposure that lasted to 12 mpf. The use of a specific inhibitor of adenosine production, the inhibitor of ecto-5'-nucleotidase (AMPCP/150 mg/kg), and the specific inhibitor of adenosine degradation, the inhibitor of adenosine deaminase, EHNA (100 mg/kg) did not affect the effects over anxiety. However, AMPCP at 3 mpf, but not EHNA, reversed aggressive parameters. AMPCP also recovered the social interaction parameter at 3 mpf in animals treated in both stages, while EHNA recovered this parameter just in those animals treated with ethanol during the gastrula stage. These results suggest that long-lasting behavioural effects of ethanol can be modulated by intervention on ecto-5'-nucleotidase and adenosine deaminase activities.

Figure 1

The locomotor parameter distance travelled (m) evaluated at 3 mpf (A) and 12 mpf (B) in animals treated with 1% ethanol during the gastrula/segmentation or pharyngula stages. AMPCP or EHNA inhibitors alone did not generate significant effects on any parameter or period of ethanol exposure when compared to the control/saline group. Data were expressed as mean ± SD and analyzed by two-way ANOVA. Multiple analysis of group was performed using Tukey’s test (n = 10 animals per group per experiments).

Figure 2

The locomotor parameter mean speed (m/s) evaluated at 3 mpf (A) and 12 mpf (B) in animals treated with 1% ethanol during the gastrula/segmentation and pharyngula stages. AMPCP and EHNA inhibitors alone did not generate a significant effect on any parameter or period of ethanol exposure when compared to the control/saline group. EHNA inhibitors had a mild effect in mean speed, when used at 3 mpf animals treated with ethanol in the pharyngula stage (A), in comparison to the ethanol/saline treated animals (*P < 0.05). (B) No effect of ethanol treatment was detected at 12 mpf. Data were expressed as mean ± SD and analyzed by two-way ANOVA. Multiple analysis of group was performed using Tukey’s test (n = 10 animals were randomized assigned for experiments).

Figure 3

Long-term effects of 1% ethanol treatment during gastrula/segmentation or pharyngula stages on the time spent in the upper zone (seconds) at 3 mpf (A) and 12 mpf (B). At 3 mpf, ethanol increased the time spent in the upper zone for all groups (*P < 0.05; **P < 0.01; ***P < 0.001) in comparison to control group (water/saline). AMPCP or EHNA inhibitors alone did not generate a significant effect on any parameter or period of ethanol exposure when compared to the control/saline group. Time spent in the upper zone was registered for 5 min by video recording in the tank diving behavioural test. Data were expressed as mean ± SD and analyzed by two-way ANOVA for 3 mpf and one-way ANOVA for 12 mpf. Multiple analysis of group was performed using Tukey’s test (n = 10 animals were randomized assigned for experiments).

Figure 4

Long-term effects of 1% ethanol treatment during gastrula/segmentation or pharyngula stages on aggressive behaviour at 3 mpf (A) and 12 mpf (B). Treatment with ethanol during the gastrula/segmentation stage and the use of AMPCP or EHNA inhibitors alone did not generate a significant effect compared to the control/saline group. (A) At 3mpf, the 1% ethanol treatment during pharyngula stage caused an increase in aggressive behavior in comparison to control group (water/saline) (*P < 0.05) and ethanol-treated animals at gastrula/segmentation stage receiving saline (##P < 0.01). This effect was recovered by AMPCP at 3 mpf (##P < 0.01). (B) At 12 mpf, the 1% ethanol treatment during pharyngula stage caused an increase in aggressive behavior in comparison to control group (water/saline) (*P < 0.05) and ethanol-treated animals at gastrula/segmentation stage receiving saline (#P < 0.05). Data were expressed as mean ± SD and analyzed by two-way ANOVA. Multiple analysis of group was performed using Tukey’s test (n = 8–10 animals were randomized assigned for experiments). Zone 1 = the nearest zone to the mirror.

Figure 5

Long-term effects of 1% ethanol treatment during gastrula/segmentation or pharyngula stages on social interaction behaviour at 3 mpf (A) and 12 mpf (B). The use of AMPCP or EHNA inhibitors alone did not generate a significant effect on any parameter or period of ethanol exposure when compared to the control/saline group. (A) At 3 mpf, the treatment with ethanol during the gastrula/segmentation and pharyngula stages decreased the social interaction (**P < 0.01; ****P < 0.0001) in comparison to control group (water/saline). These effects were recovered by AMPCP and EHNA in the gastrula/segmentation stage and by AMPCP in the pharyngula stage at 3 mpf. (B) No effect of ethanol treatment persisted until 12mpf. Data were expressed as mean ± SD and analyzed by two-way ANOVA for 3 mpf and one-way ANOVA for 12 mpf. Multiple analysis of group was performed using Tukey’s test (n = 10 animals were randomized assigned for experiments). Stimulus side = the side of aquarium near to the aquarium containing 15 conspecific fish.