| Objective:The health damage caused by environmental Lead（Pb）pollution has been a global public health focus,especially the effect of Pb on the learning memory and cognitive function.Hippocampus is the structural basis of learning,memory and cognitive function,and the synaptic plasticity is the biological basis of learning memory and cognitive.Therefore this study establishes different doses,different periods and patterns of Pb exposure rat models to explore the relationship among changes of learning and memory function in Pb exposure rats,changes of morphology of dendritic spines in hippocampal trisynaptic circuit,and changes of the number of postsynaptic AMPA receptor,which can provides a theoretical basis and foundation to illuminate the role of synaptic plasticity changes in the damage of learning and memory function to Pb exposure.Methods:We selected 9 weeks sexually mature male and female SD rats according to 2:1cage match,then the successful pregnant female rats were randomly assigned to control group（only drank deionized water）,500 mg/L lead acetate and 2000 mg/L lead acetate exposure groups(which were equivalent to 319 and 1275 mg/L Pb2+),and the way of Pb exposure was via drinking water,then until the offspring weaned,that is,3 weeks after birth（postnatal week 3,PNW3）;The group of male offspring corresponded with female group,the groups contained control,low lead exposure group（LLG）and high lead exposure group（HLG）,respectively drinking 0 mg/L,100 mg/L and 500 mg/L lead acetate water solution(LLG and HLG is 64 mg/L and 319 mg/L Pb2+).In addition,we set the high to control lead exposure group（HCLG）,that is,after these corresponding male offspring rats weaning,whose female drank 2000 mg/L Pb aqueous solution,then fed normal and no longer expose to Pb,and these rats reared to 52 weeks after birth（postnatal week 52,PNW52）.When the SD rats were in PNW3 and PNW52,we used the inductively coupled plasma atomic emission spectrometer（ICP-OES）to detect Pb content in blood,cortex and hippocampus.Morris water maze Analysis System was applied to detect spatial learning and memory of the rats.Golgi-Cox staining method（Golgi-Cox）was conducted to observe the changes of number and morphology of dendritic spines in hippocampal CA1,CA3,DG area and entorhinal cortex.The transmission electron microscopy was used to observe the changes of number and morphology of synapses in hippocampal CA1.And we used confocal laser scanning microscope（CLSM）and immunofluorescence double labeling of PSD-95 and GluR1 protein to detect the changes of protein expression and the co-localization of PSD-95 and GluR1 protein.Results:1.Effect of Pb content in blood,cortex and hippocampus of SD rats by different different dose,time and model of Pb exposure:ICP-OES detection showed that in PNW3 and PNW52 rats,the Pb contents of blood,cortex and hippocampus were increased with the increase of Pb exposure concentration and time,presenting a dose-response relationship（P<0.01）;In addition to the control group was not detected out,the Pb content in blood,cortex and hippocampus was increased with the increase of age（P<0.01）;In HCLG,Pb content in blood,cortex and hippocampus was lower than the detection limit,basically the same with the control.2.Effect of learning and memory in SD rats by different dose,time and model of Pb exposure:In Morris water maze navigation experiment,for PNW3 and PNW52 rats,since the training of third or fourth days,the average escape latency presented varying degrees of extension in each Pb exposure group compared with the control（P<0.05）,In spatial probe test,the times of crossing the platform decreased in Pb exposure group,and there was statistically significant difference compared with control group（P<0.05）.For the PNW52 rats,compared with the PNW3 rats,the average escape latency and the times of crossing the platform had no statistical significance（P>0.05）.The time of finding platform to escape latency decreased in HCLG compared with the HLG,but still higher than the control,and the difference had no statistical significance.And the number of crossing platform had not significantly improved,and was still lower than the control,the difference is statistically significant（P<0.05）.3.Effect of synaptic plasticity of dendritic spines structure of the hippocampal circuit in SD rats by different dose,time and model of Pb exposure:Golgi-Cox staining detected that for PNW3 and PNW52 rats the spine density had different degrees of reduction in rat hippocampal trisynaptic circuit（EC,DG,CA3,CA1）with the increase of Pb concentration（P<0.05）.Pb exposure made the percentage of dendritic spines filiform pseudopodia of hippocampus synaptic circuitry and the percentage of slender dendritic spine in hippocampal CA1 and DG decrease in different ages.At the same time,Pb exposure induced the mushroom type dendritic spine ratio decrease in CA1,DG and EC of PNW3 rats,and CA1 and CA3 regions of PNW52 rats.And the thick short spines ratio increased in hippocampal synaptic circuits of different ages except CA3 of PNW52 rats（P<0.05）;The dendritic spines density of control SD rats in the PNW52 period was larger than in the PNW3 period,and for the DG region of PNW52Pb exposed group,the dendritic spine density was basically consistent with the PNW3period,the difference was not statistically significant（P>0.05）.For control group of hippocampal synaptic circuit district,the percentage of filopodia spines in PNW52 rats compared with PNW3 rats decreased significantly,and in Pb exposure groups,the percentage of filopodia spines in PNW52 rats compared with PNW3 rats decreased significantly,the difference is statistically significant（P<0.01）.And in addition to CA3,the trend change of slender dendrites spines in the other areas at different time was opposite to the trend change of the pseudopodia silk dendritic spine;Comparing the HCLG with HLG,except dendritic spines density in the DG area improved,in other districts between the two groups,the difference had no statistical significance（P>0.05）.And,compared with the control,the dendritic spine density was still significantly reduced（P<0.05）.In HCLG,each region of hippocampal synaptic circuit,the filopodia spines ratio declined,compared with control,and no change compared with HLG.The slender dendritic spines ratio of hippocampal CA1 and DG in HCLG declined,compared with control group,no change compared with HLG,and the mushroom spines ratio of hippocampal CA1,CA3 and entorhinal cortex in HCLG declined,compared with control group,no change compared with HLG,while the stubby dendritic spines ratio of hippocampal CA1,CA3 and DG in HCLG increased,compared with the control,no change compared with HLG.4.Effect of synaptic structural plasticity in SD rats by different dose,time and model of Pb exposure:Electron microscopy detected,Pb exposure caused the number of hippocampal synaptic reduce and postsynaptic density PSD thickness thin in PNW3rats,also made the number of hippocampal synaptic reduce,the width of synaptic cleft increase and curvature of the synaptic interface decrease in PNW52 rats（P<0.05）;with the increase of age,the thickness of PSD became thinning,the synaptic cleft became narrow,and the PNW52 rats of LLG compared with corresponding PNW3 rats,the thickness of PSD reduced（P<0.01）.The number of synapses and curvature of synaptic interface showed no change by time,the number of synapses in PNW52 rats of HLG was significantly lower than in PNW3 rats;compared with the HLG,damage effects in the number of synapses,the thickness of PSD,the synaptic cleft,length of synaptic active zone and curvature of the synaptic interface were somewhat improved,however,in addition to the number of synapses increased with significant difference（P<0.05）,other difference had no statistical difference.5.Effect of synaptic functional plasticity in SD rats by different dose,time and model of Pb exposure:Immunofluorescence double staining found that,Pb exposure induced the reduction of PSD95 expression in CA1 region of PNW3 rats and in EC region of PNW52 rat（P<0.05）.Expression of GluR1 in CA3 of PNW52 rats and trisynaptic circuit districts except CA1 of PNW3 rats decreased after Pb exposure（P<0.05）.In addition to the EC area,in Pb exposure group,the PSD-95-GluR1 co localization point in other districts of PNW3 rats increased,compared with the control（P<0.05）.The expression of PSD95 in CA1 regions of control and LLG as well as DG and EC region of HLG in PNW52 rats declined,compared with corresponding PNW3rats（P<0.05）.The expression of GluR1 in CA3 of control,all synaptic loop area of LLG,and other areas except the CA3 of HLG in PNW52 rats also declined,compared with corresponding PNW3 rats（P<0.05）.PSD-95-GluR1 co localization point in all synaptic circuit area of control and DG area of HLG in PNW52 rat increased,compared with corresponding PNW3 rats（P<0.05）;and CA3 area of HLG decreased（P<0.05）;compared with the control,in HCLG we found the decrease of PSD95 expression in EC area and the increase of GluR1 expression in CA3 region.And compared with the control,in HCLG PSD-95-GluR1 co localization point increased in the hippocampal CA3,and compared with HLG,the PSD-95-GluR1 co localization point in hippocampal CA1 and DG regions appeared to reduce in HCLG,while to increase in EC region.Conclusion:1.In this study,through establishing rat model of nerve injury induced by Pb exposure of different doses,time and modes,we observed that impairment of learning and memory ability in rats induced by Pb exposure was dose-dependent,and the early age of development to Pb exposure,after weaning Pb exposure was no longer given,the nervous system damage was hard to natural recovery.2.This study applied Golgi-cox and transmission electron microscopy（TEM）,and found that Pb exposure can Pb to rat hippocampal trisynaptic circuit of synaptic structural plasticity abnormal,which showed in the abnormal of rat dendritic spine and synapse number,morphology and structure.3.This study firstly found that Pb exposure can Pb to the synaptic functional plasticity change of the rat hippocampal trisynaptic circuit districts about GluR1expression in the PSD localization by immunofluorescence and laser confocal technique. |
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