The Role Of MTOR In The Neurobehavioral Impairment Of Zebrafish Larvae Induced By Nano-alumina

Objective:The present study aims to introduce zebrafish larval model to neurobehavioral impairment induced by Nano-alumina and to investigate the role of m TOR in the neurobehavioral impairment.Methods:The 6hpf zebrafish embryos were randomly divided into 6 groups and exposed to various concentrations of Al NPs suspensions(0,12.5,50,100,200μg/m L)for 6 days until6 dpf.Embryos were transferred into 6-well plates,20 embryos per well and 160 embryos per group.Al NPs suspensions were changed daily for stable concentrations.A total of 8plates were used to observe the neuromotor behavior alternations.The neuromoter behavior of zebrafish Laval in early developmental stage was determined by tracking the motor behavior and thigmotaxis reaction under dark and bright shifting stimulus.Aluminum contents in the heads of zebrafish larvae were measured by ICP-MS.The expression of m TOR protein and gene in the larval heads were detected by the whole-mount IHC and RT-PCR,respectively.In addition,autophagy-related genes under the m TOR pathway were monitored.Furthermore,the expression of m TOR gene was specially knocked-down to verify the role of m TOR in the early neuromotor behavior alternations of zebrafish larvae.Results:1.The results of neuromoter behavior alternations in zebrafish larvae treated by Al NPs: Compared with the solvent control group,the movement speeds and distances traveled by the larvae in 25μg/m L group were significantly increased(P<0.05).However,those in 50μg/m L group showed a trend of decline.There was a significant decrease(P<0.05)of the thigmotaxis degree in 200μg/m L group than that in solvent control group.The panic and avoidance reflex in 100μg/m L group and 200μg/m L group were both significantly increased(P<0.05)than that in the control group.2.The results of aluminum contents in the heads of larva: The color of the embryos and the heads of larvae were both darken after 24 h exposure of Al NPs,especially in the 100μg/m L group.Compared with the solvent controls,aluminum content in the heads of larva in the 100μg/m L group was significantly increased(P<0.05).3.The results of RT-PCR: The gene expression of m TOR in 50,100,200μg/m L Al NPs treated groups was significantly decreased than that in the control group(P<0.05).The gene expression of AKT3 in 25,50μg/m L Al NPs treated groups was significantly decreased than that in the control group(P<0.05).The gene expression of ULK1 in 50,100,200μg/m L Al NPs treated group was significantly increased than that in the control group(P<0.05).The gene expression of ULK2 in 100,200μg/m L Al NPs treated group was significantly increased than that in the control group(P<0.05).The gene expression of Beclin1 in 200μg/m L Al NPs treated group was significantly increased than that in the control group(P<0.05).4.The results of the whole-mount IHC: Compared with the solvent control group,the fluorescence signals decreased in the heads of larvae in 50,100,200μg/m L groups,indicating that the expression of m TOR protein was down-regulated with the increase concentrations of Al NPs.5.The results of Microinjection: There was no significant difference in the movement speed and distance between the microinjection group and the control group(P>0.05).However,there was a significant decrease in the thigmotaxis of larvae in microinjection group compared with that in the control group(P<0.05),the panic reflex ability of larvae in microinjection group was significantly decreased as well(P<0.05).The gene expression of m TOR in microinjection group was significantly decreased(P<0.05).Conclusion:1.Nano-alumina mainly affects the thigmotaxis and the panic-avoidance reflex of zebrafish larvae,there is a low dose excitation effect on motor behavior.2.Nano-alumina can accumulate in the heads of larvae and the contents coorespond to the treated concentrations of Al NPs.3.The protein and gene expression of m TOR in the larval head significantly decreased in Nano-alumina treated groups.4.The specific knockdown of m TOR gene can impair the early neurodevelopmental behavior of zebrafish larvae.