| Children cognitive impairment caused by lead exposure during development stage is one of the most important health problems all over the world.The mechanism underlying lead induced neurotoxity is one of research hotspots in the area of neurotoxicology.The impact of lead on synaptic structure and function is manifested as: structural alteration in the morphology and density of dendritic spines,and functional changes in synaptic transmission and plasticity.The structural and functional changes of synapse induced by lead exposure further impaired the efficiency of information transmission in neural network,finally causing cognitive decline.Therefore,it has important theoretical and practical significance to clarify the mechanism underlying lead induced synaptic changes.Calcium ion is an important second messenger in neuron.It participates in synaptogenesis,and structural and functional changes of synapses.The calcium channels expressed in neuronal surface include voltage gated calcium channel(VGCC),receptor operated calcium channel(ROCC)and transient receptor potential(TRP).Such channels have different expression levels and properities,which are associated with synaptic changes.Previous studies demonstrated that lead ion disrupted calcium homeostasis and its biological function.Our previous study indicated that lead exposure caused decrease in hippocampal dendritic spines density and synaptic plasticity.Meanwhile,the calcium current also decreased.The mechanism underlying these phenomenons also deserves to be explored.Cold-inducible RNA binding protein(CIRBP)plays significant roles in neurophysiology,such as proliferation,apoptosis,blood brain barrier maintaince and glucose metabolism.Recent studies revealed that CIRBP was able to modulate expression of multiple ion channels.Our previous results indicated that CIRBP protein was downregulated by lead exposure.CIRBP overexpression induced thioredoxin upregulation thus protecting neuron from oxidative stress injury.Meanwhile,bioinformatics study indicated that CACNB3 and CACNA1 A were downregulated after CIRBP interference.This study suggested that CIRBP might modulate calcium channel function through regulating CACNB3 and CACNA1 A expression.Thereby,the current study focused on the alteration of neuronal calcium signal and CIRBP expression.We recorded the real-time calcium activity in living neuron,aiming at clarifying the effect of lead on neuronal calcium activity and the role of CIRBP on such process.In a word,we provided some experimental evidences for the mechanism underlying lead induced synaptic changes,which may help to protect neural system from lead toxity.Aims:The current study tried to find out lead exposure induced changes in synaptic structure,function and cellular calcium signal.The current study tried to clarify the role of CIRBP protein in such processes,aiming at providing a basis for lead exposure induced neurological impairment.Methods:1.To establish lead exposure animal model,SD rats were exposed to lead acetate solution(0 ppm or 300 ppm)as drinking water for 8 weeks.After exposure,the body weight of each group was measured.The blood lead concentration was measured by an atomic absorption spectrophotometer.Morris water maze was employed to examine the effect of lead exposure on spatial learning and memory capacity in rats.2.To establish lead exposure model in vitro,the hippocampus of embryonic day 16~18 rat embryos were dissected out for primary neuronal cell culture.Lead exposure(5 μM)started at day 7 in vitro and the primary neurons were cultured until day 24 in vitro.The examinations were conducted following experimental design.3.Electrophysiological methods were employed to measure the alteration in long-term potentiation(LTP)、miniature excitatory postsynaptic current(m EPSC)and firing numbers of action potentials(AP)after lead exposure or CIRBP KO.4.Laser confocal microscopy,live cell imaging system and lentivirus transfection were combined to observe the dendritic spines and synchronous spontaneous calcium oscillation in cultured primary neurons.5.Western blot was used to detect expression of CIRBP,CACNA1 A and CACNB3.Immunofluorescence was employed to test CIRBP and microtubule associated protein 2(MAP2)proteins expression in brain slices and cultured neurons.6.RNA binding protein immunoprecipitation(RIP)and PCR methods were employed to explore that if CIRBP protein could bind to CACNB3 m RNA.Results:1.Lead exposure induced impairment in synaptic structure and function1)Lead exposure did not cause differences in body weight of rats.The lead-exposed rats had significantly higher blood lead concentration than the control rats(P<0.05).2)The field excitatory post-synaptic potential(f EPSP)slopes of LTP was significantly lower in lead-exposed rats than the control rats(P<0.05).3)The cultured primary neurons presented intact morphological and much branches.The neuronal marker MAP2 was expressed in more than 90% of cultured neurons.Lead exposure caused significant decrease in density of dendritic spines,frequency and amplitude of m EPSC(P<0.05).The firing rate of AP was also downregulated by lead exposure(P<0.05).2.Lead exposure induced alteration in synchronous spontaneous calcium oscillation in cultured primary neurons1)The current study successfully detected synchronous spontaneous calcium oscillation in cultured primary neurons.As the time flows,the frequency of calcium oscillation gradually rised and then gradually decreased,with a peak in day 16 in vitro.The amplitude of calcium oscillation fluctuated in a small range.2)The calcium oscillation in lead-exposed primary neurons was similar with control neurons with an except of significant lower frequency in day 16 in vitro(P<0.05).3)The calcium oscillation disappeared when the normal culture medium was changed to calcium-free medium.The calcium oscillation recoverd when changed to normal culture medium.3.The role of CIRBP protein in the effect of lead on calcium oscillation and synaptic changes1)CIRBP expression was significantly downregulated by lead exposure in SD rats and primary neurons(P<0.05).2)Genetic sequencing indicated that the CIRBP gene was exactly mutated in our previously constructed CIRBP KO rat.The brain sections of CIRBP KO rat had no CIRBP protein expression.3)CIRBP KO rats had lower body weight than wild type rats(P<0.05).The swimming speed of CIRBP KO rats was similar with wild type rats.The CIRBP KO rats had significantly less times of crossing the platform in testing phase(P<0.05).The f EPSP slopes of LTP was significantly lower in CIRBP KO rats than wild type rats(P<0.05).4)CIRBP KO caused significant decrease in density of dendritic spines,frequency of m EPSC and the firing rate of AP(P<0.05).CIRBP KO downregulated frequency of calcium oscillation in day 14,16,18 in vitro(P<0.05).Moreover,CIRBP KO downregulated CACNB3 protein expression in primary neurons(P<0.05),while had no obvious effect on CACNA1 A expression.5)Lead exposure had no obvious effect on CACNA1 A protein expression while downregulated CACNB3 protein expression.6)CIRBP overexpression(OE)by lentivirus transfection significantly upregulated CIRBP and CACNB3 protein level in primary neurons(P<0.05).Moreover,CIRBP OE partialy restored lead exposure induced downregulation in CACNB3 protein level,frequency of calcium oscillation and density of dendritic spines(P<0.05).7)CIRBP protein may regulate CACNB3 protein expression through binding to its 3’ UTR.Conclusions:1.Lead exposure during development stage impaired spatial memory ability of rat and synaptic plasticity of CA1 region.Lead exposure also induced decrease in density of dendritic spines and excitability of hippocampal neurons.2.As the time flows,the frequency of calcium oscillation gradually rised and then gradually decreased with a peak at day 16 in vitro.Lead exposure decreased the frequency of calcium oscillation at day 16 in vitro.3.CIRBP downregulation participated in lead induced changes in calcium oscillation and synaptic structure and function.The binding of CIRBP protein to CACNB3 m RNA may be the mechanism underlying above changes. |
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