Effects Of Exogenous Lead Exposure On Synaptic Transmission Of Primary Hippocampal Neurons And Its Possible Mechanism

Background and purpose: Food lead poisoning incidents is gaining more and more attention.A large number of studies have shown that lead can lead to brain damage to the nervous system,and thus affect the learning and memory and cognitive ability,of which the development of children’s injury is particularly evident.The transmission of synaptic neurotransmitters is the basis for maintaining the normal work of brain activity,however,the effect of lead on the transmission of neurotransmitters and its mechanism are not clear.Therefore,the purpose of this study is to investigate the effects of chronic lead exposure on the synaptic transmission of primary hippocampal neurons and its possible mechanism.Methods: 1.Sprague-Dawley(SD)neonatal rats(within 24 hours of birth)ware used.The hippocampal CA1 tissue was cultured in vitro and 5μM of lead acetate was added on the third day for chronic lead exposure.On day 14,miniature excitatory postsynaptic currents(m EPSC)and miniature inhibitory postsynaptic currents(m IPSC)were recorded by using electrophysiological patch-clamp technique to evaluate the effect of chronic lead exposure on excitatory synaptic transmission and inhibitory synaptic transmission.2.SD rats were chosen as the research model.SD rats were exposed to 250 parts permillion(ppm)lead acetate during pregnancy to the first 30 days of birth.The distribution of synaptic membrane vesicles in CA1 tissue was observed by projection electron microscopy;The m RNA expression of SNARE monomer syntaxin1 a,SNAP-25 and VAMP2 was detected by Q-PCR;Western Blot was used to detect the protein expression of SNARE complex;Electrophysical patch clamp technique for recording paired-pulse ratio(PPR).On the other hand,in order to elucidate the possible mechanism of the effect of chronic lead exposure on synaptic transmission,the primary cultured hippocampal neurons were modeled exposed by 5μM lead for 3 to 14 days.1.Western protein was used to detect the protein content of H3K27me3,synapsin1 and the phosphorylation levels of synapsin1.2.The H3K27me3 interaction with synapsin1 were detected by chromatin immunoprecipitation assay.3.The m RNA expression of synapsin1,synapsin2,synaptotagmin6 and CDK5 in H3K27me3 downstream regulatory was detected by Q-PCR.4.Electrophysiological patch clamp technique further confirms the presence of CDK5 in the inhibition of the release of neurotransmitters caused by chronic lead exposure.Results: 1.Chronic lead exposure significantly reduced the frequency of m EPSC and m IPSC in primary hippocampal neurons,but there was no significant change in m EPSC and m IPSC amplitude.2.The results of transmission electron microscopy showed that chronic lead exposure resulted in more diffuse distribution of presynaptic membrane vesicles in presynaptic menbranes.Q-PCR results showed that chronic lead exposure resulted in a significant decrease in the m RNA levels of SNAP-25 and VAMP2,a significant increase in the m RNA content of syntaxin1 a.However,the results of Western Blot showed that the SNARE complex protein content remained unchanged.3.Chronic lead exposure resulted in a significant decrease in protein content of H3K27me3.Gene related to vesicle release was screened by gene chip technology.Q-PCR results showed that chronic lead exposure significantly reduced synapsin1 m RNA levels,but synapsin2 levels were significantly increased.The results of the Chip test showed that the binding capacity of H3K27me3 and synapsin1 was increased in the chronic lead exposure group.4.Chronic lead exposure significantly reduced the phosphorylation level of tryptophan site at position 553 of synapsin1,and did not change the total protein content of synapsin1.Chronic lead exposure significantly increased the expression of CDK5 m RNA in the upstream of the synapsin1 phosphorylation site.Conclusion: 1.Chronic lead exposure inhibits the release of excitatory and inhibitory neurotransmitters in primary hippocampal neurons,but does not alter the current intensity of the postsynaptic membrane.2.Chronic lead exposure changes the distribution of synaptic membrane vesicles and inhibits the release of presynaptic membrane vesicles.3.Chronic lead exposure increases the expression of CDK5,resulting in the phosphorylation of the first 553 tryptophan site of synapsin1 and thus the release of neurotransmitters.