Progressive Impairment Of Learning And Memory In Adult Zebrafish Treated By Al₂0₃ Nanoparticles When In Embryos

Objective:Al₂O₃ nanoparticles（Al₂O₃-NPs）,a material with excellent physical and chemical properties,have been widely used in the fields of optoelectronics,catalysts,engineering,medicine and other fields.Al₂O₃-NPs can be released into the environment directly or indirectly during product manufacturing,processing and waste discharge.The Al₂O₃-NPs particles may induce toxic effects when they are accumulated in the organism to a certain threshold.However,the existing toxicological evidence makes it difficult to accurately assess the potential risks of Al₂O₃-NPs to aquatic organisms and the human body.Therefore,this study aims to investigate the effect of Al₂O₃-NPs on learning and memory of adult zebrafish.Methods:Six concentrations（0,6.25,12.5,25.0,50.0,and 100 mg/L）of Al₂O₃-NPs at 13nm particle size were administrated to zebrash embryos after 6 hours post-fertilization for 6 days.Learning and memory abilities and different biochemical indicators were tested at adulthood（6,9 and 12 months of age）.This study designed a set of experiments to assess whether zebrafish embryonic exposure to Al₂O₃-NPs can accumulate in brain tissue and impair its learning and memory abilities.We observed the characterization of Al₂O₃-NPs with an electron microscope and measured the Zeta potential with Al₂O₃-NPs size analyzer.Anxiety activity test,exploratory activity test,and T-maze test were used to observe the changes in neurobehavior and learning and memory of adult zebrafish at 6,9 and 12 months of age.In addition,neurotransmitter biomarkers such as acetylcholine（ACh）,dopamine（DA）,and oxidative stress markers such as reactive oxygen species（ROS）and lactate dehydrogenase（LDH）were measured.Additionally,the changes in the number of nerve cells in brain tissue were detected by Nissl staining,and the pathological changes in brain tissue were observed by electron microscopy.RT-PCR detected the expression of autophagy-related genes（ULK1,LC3-Ⅱ,Beclin-1）.The accumulation of aluminum in brain tissue was detected by ICP-MS.Results:（1）After exposure to Al₂O₃-NPs in zebrafish embryos,the aluminum content in brain tissue increased at 6 and 12 months of age,with a dose-dependent effect.（2）Zebrafish embryos were exposed to Al₂O₃-NPs,and anxiety-like behavior increased at 6,9 and 12 months of age.Including the average speed and the percentage of accumulated time in the outer zone decreased significantly,and the freezing time movement rate increased.There was a dose-and age-dependent effect.As the dose and age increased,anxiety-like behaviors gradually increased.（3）The exploration behavior of the new environment is weakened:At 6,9 and12 months old,the exploration behavior of the zebrafish is weakened when the embryo is exposed to Al₂O₃-NPs,and there is a dose-and age-dependent effect.With the increase of dosage and age,the exploration behavior gradually weakened.（4）Exposure of Al₂O₃-NPs to zebrafish embryos resulted in reduced learning and memory abilities of 6-and 12-month-old zebrafish,and there were dose and age-dependent effects,including increased latency and reduced cumulative residence time.As the dose and age increased,learning and memory abilities progressively deteriorated.（5）Exposure of Al₂O₃-NPs to zebrafish embryos reduced the levels of neurotransmitters in the brain tissue of 6-and 12-month-old zebrafish,including a significant decrease in the levels of acetylcholine（ACh）and dopamine（DA）.（6）Exposure of Al₂O₃-NPs to zebrafish embryos resulted in aggravated oxidative stress responses in brain tissues of 6-and 12-month-old zebrafish,including significant increases in reactive oxygen species（ROS）and lactate dehydrogenase（LDH）levels.（7）Exposure of Al₂O₃-NPs to zebrafish embryos leads to a decrease in the number of nerve cells in the brain tissue of zebrafish and the occurrence of autophagy,and the expression of autophagy-related genes（ULK1,LC3-Ⅱ,Beclin-1）was up-regulated in brain tissue..Conclusion:Al₂O₃-NPs have neurotoxicity to zebrafish in a fairly wide concentration range of 6.25 mg/L to 100 mg/L.We found that the low concentration（6.25 mg/L）Al₂O₃-NPs dosage is relative safer to the zebrafish before 6 months-old age.With the increase of exposure dose and age,all dosages of Al₂O₃-NPs including the low concentration（6.25 mg/L）can induce zebrafish learning and memory impairment.Exposure to Al₂O₃-NPs in zebrafish embryonic has a dose and age-dependent effect on the aluminum content in zebrafish brain tissue,and it increases significantly with increasing dose and age.Exposure to Al₂O₃-NPs can cause progressive neurobehavioral changes and progressive impairment of learning and memory abilities.It also causes oxidative stress and dopaminergic transmission in the zebrafish brain tissue to be damaged,and the number of nerve cells in the brain tissue is significantly reduced and accompanied by autophagy.