| Objective: Environmental heavy metal pollution has become a world public health problem that seriously affects people’s health.Heavy metals of lead(Pb),cadmium(Cd)and mercury(Hg)were the mainly reported that could cause serious health effect.These heavy metals often coexist in the environment and enter human body through various ways.Thus,all these heavy metals could be detected in human blood sample at the same time.Our previous studies have found that combined exposure to heavy metals of Pb,Cd,and Hg at the blood load level of general population caused learning and memory impairments in rats.However,the mechanisms remain unclear.N-methyl-Daspartate(NMDA)receptors-dependent synaptic plasticity plays an important role in learning and memory.With this in mind,we here explored the role of NMDA receptors in learning,memory,and synaptic plasticity damage caused by low-dose combined exposure to heavy metals of Pb,Cd,and Hg by establishing exposure rat,primary cultured hippocampus neurons,and acutely isolated slice models.Our study will provide a new clue for prevention and treatment of neuronal system impairment caused by low-dose heavy metals combined exposure.Methods: 1.The effect of low-dose heavy metal mixture exposure on the NMDA-m EPSCs and the expression of NMDA receptors Primary cultured hippocampal neurons were exposed to single heavy metal,two heavy metal mixtures,and three heavy metal mixtures of Pb,Cd,and Hg with gradient concentrations,respectively.The NMDA mediated mini excitatory postsynaptic currents(NMDA-m EPSCs)were recorded by using patch clamp with specific inhibitors.In addition,the expression of NMDA receptors subunits(NR1,NR2 A,and NR2B)was assessed with western blotting.2.The effect of low-dose heavy metal mixture acute perfusion on f EPSP in rat brain slice model.Using the brain patch clamp,the schaffer collateral field excitatory post synaptic potential(fEPSP)slope ratio of the hippocampus in rat brain slices was recorded before and after concentration gradient of heavy metal Pb,Cd,and Hg mixtures perfusion.3.The effect of low-dose heavy metal mixture exposure on learning and memory,LTP,and NMDA receptors subunits expression in the rat model.Adult SD rats were paired with the ratio of female: male was 2:1.The conceived females were then randomly divided into groups of Control gourp(drinking distilled water),1×Pb+Cd+Hg group(drinking water containing heavy metal mixtures of 20.0 mg/L lead acetate trihydrate,7.0 mg/L cadmium chloride,and 0.1 mg/L mercuric chloride),5×Pb+Cd+Hg group(the concentrations of heavy metal mixtures in drinking water were five times of 1×Pb+Cd+Hg).The male offspring of postnatal day 21(PND21)were then used to conduct experiments after prenatal and lactational heavy metals exposure.The Pb and Cd concentrations in blood and brain were assayed by using inductively coupled plasma mass spectrometry(ICP-MS),while Hg levels in blood and brain were detected with cold vapor atomic fluorescence spectrometry(CVAFS).Learning and memory abilities of rats were measured by the Morris water maze test.The induction of long-term potentiation(LTP)in rat hippocampus was recorded by patch clamp.The structure and neuronal morphology of rat hippocampal were analyzed by hematoxylin-eosin(HE)staining.The density and morphology of dendritic spines in each region of rat hippocampus were observed by Golgi-cox staining.The protein and gene expression levels of NMDA receptors subunits(NR1,NR2 A,and NR2B)were detected by western blotting and PCR.Results: 1.The effect of low-dose Pb,Cd,and Hg mixtures exposure on the NMDAm EPSCs in primary cultured hippocampal neurons.The NMDA mediated m EPSCs was recorded in the model of primary cultured hippocampal neurons by using patch clamp with specific receptors inhibitor.When compared to Control,the amplitude of m EPSCs was decreased in a concentrationdependent manner after exposure to three heavy metal mixtures of Pb,Cd,and Hg,and the significantly difference was found between Control and 5×Pb+Cd+Hg groups(P<0.05).However,no significant differences in m EPSCs were found among groups of 5×Pb,5×Cd,5×Hg,5×Pb+Cd,5×Pb +Hg,5×Cd+Hg,and Control.These results suggested that combined exposure to Pb,Cd,and Hg could synergy induce synaptic neurotoxicity and inhibit NMDA receptor-mediated synaptic transmission,even the individual heavy metals are in a level that lower than no observed adverse effect levels(NOAELs).2.The effect of low-dose Pb,Cd,and Hg mixtures exposure on the NMDA receptors subunits in primary cultured hippocampus neurons.The expression levels of NMDA receptors subunits in primary culture of hippocampal neurons were detected by Western blotting.The protein expression levels of NR1 and NR2 A decreased in a concentration-dependent manner after exposure to Pb,Cd,and Hg,and the significant difference was found between groups of 1×Pb+Cd+Hg(or 5×Pb+Cd+Hg)and control(P<0.05).However,there was no significant difference in NR2 B protein expression between these exposed groups and the Control group(P>0.05).3.The effect of acute Pb,Cd,and Hg mixtures exposure on hippocampus f EPSP in rat brain slices.The f EPSP slope ratio of hippocampus Schaffer collateral was detected in brain patch clamp.When compared to Control,the f EPSP slope ratio of hippocampus Schaffer collateral was decreased with the increased concentration of heavy metals mixtures,and the significant difference started to appear from 15×Pb+Cd+Hg(P<0.05).However,there was no significant change in single heavy metal exposure groups with the same exposure levels as 15×Pb+Cd+Hg goup(P>0.05).4.The effect of low dose Pb,Cd,and Hg mixtures exposure on LTP in rat hippocampus.Using ICP-MS and CV-AFS,we found the contents of Pb,Cd and Hg in rat blood and brain tissue increased in a dose-responsive manner when exposure to increased heavy metal mixtures(P<0.01).In addition,the blood Pb,Cd,and Hg level of rats in 1×Pb+Cd+Hg group were 28.03 ± 4.26 μg/L,0.63 ± 0.14 μg/L,1.38 ± 0.05 μg/L,respectively.These waere close to blood heavy metals load(Pb 34.90 μg/L,Cd 0.49 μg/L,Hg 1.81 μg/L)of Chinese general population.Using the patch clamp,we found that the LTP slope in 5×Pb+Cd+Hg exposure group was lower than that of Control after 30 min of LTP induction.The LTP slopes in groups of 1×Pb+Cd+Hg and 5×Pb+Cd+Hg were significantly decreased when compared to Control(P<0.05).Here,the results revealed that combined exposure to Pb,Cd,and Hg could induce functional dysregulation in synaptic plasticity of rat hippocampus.5.The effect of low dose Pb,Cd,and Hg mixtures exposure on spatial learning and memory in rat.The morris water maze test found that the rat escape latency was prolonged when exposure to heavy metal mixtures in a concentration-dependence manner,and the difference was found between groups of 1×Pb+Cd+Hg and 5×Pb+Cd+Hg were significantly decreased when compared to Control(P<0.05).6.The effect of low dose Pb,Cd,and Hg mixtures exposure on the morphology of rat hippocampus.In our recently exposure level of heavy metal mixtures,the structure and neurons morphology in hippocampus were observed normal.In addition,the typical pyramidal cell layer in CA1 and CA3 areas,and granular cell layer in DG area were all found without obvious difference.7.The effect of low dose Pb,Cd,and Hg mixtures exposure on density and morphology of dendritic spines in rat hippocampal neurons.Using Golgi-cox staining,we found the dendritic spine density in CA1 of 1×Pb+Cd+Hg and 5×Pb+Cd+Hg was significantly lower than that of Control(P<0.05).The dendritic spine density in CA3 of 5×Pb+Cd+Hg was also significantly lower than that of control(P<0.05).In addition,the types of hippocampus dendritic spine were changed after exposed to heavy metal mixtures.The mushroom-type and elongated dendritic spine in CA1,CA3,and DG regions decreased in a concentration-dependent manner after exposed to heavy metal mixtures(P<0.05).8.The effect of low dose Pb,Cd,and Hg mixtures exposure on the expression of NMDA receptors subunits.Using Western blotting,we found that the NR1 and NR2 A expressions were significantly started to decrease after exposed to 1×Pb+Cd+Hg compared to Control(P<0.05).However,either the NR2 B protein expression or the gene expression NR1,NR2 A,and NR2 B did not affected by the heavy metal mixtures exposure(P>0.05).Conclusions: 1.Heavy metals Pb,Cd,and Hg combined exposure could cause synergistic neurotoxicity and inhibit NMDA receptors-mediated synaptic transmission,even the respective individual heavy metal is in a level that lower than NOAELs.2.Low doses Pb,Cd and Hg can cause functional and structural dysregulation in synaptic plasticity of rat hippocampus in a concentration-dependent manner.3.Low dose Pb,Cd and Hg could induce learning and memory impairment in rats,which may be related to NMDA receptor-associated synaptic plasticity in hippocampal neurons. |
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