The Study Of Glutamate And Glutamate Receptor In Lanthanum Induced Learning And Memory Impairment

Objective:The rare earth elements（REEs）include 15 lanthanides as well as scandium and yttrium,which can be divided into light rare earths represented by lanthanum（La）and cerium,heavy rare earths represented by yttrium and lutetium.REEs can enter the human body through the respiratory tract,digestive tract and other channels,and accumulate in the organs,causing damage to many systems such as liver,kidney,immune,nerve and endocrine.The nervous system is very sensitive to the REEs,and its damage effect is also widely concerned.Epidemiological study has shown that children’s intellectual development levels in high-REEs regions are significantly lower than that in non-rare-earth regions.Animal experiments also confirmed that REEs could decrease the development of nervous system,neurobehavioral capacity and learning and memory ability.La is a typical and widely used REE.It has been reported that the blood La content in children lived in the REEs mining area was 2.26 times higher than the control area.Feng and other studies found that after 6 weeks of continuous intake of lanthanum chloride（lanthanum chloride,LaCl₃）（2 mg/kg BW/day）in rats,the contents of La in the hippocampus are 0.014±0.007μg/g.Daily injection of 2 mg/kg LaCl₃ in the chick’s brain for 1 week could obviously impair its long time process memory.In vitro cell tests,La could lead to mitochondrial dysfunction in primary cultured neurons of the rat,oxidative damage of astrocytes.The above studies show that La has obvious neurotoxicity.However,the potential mechanisms about the effects of La remain unclear and require further exploration.Glutamate（Glu）is an important neurotransmitter in the central nervous system,which plays a key role in the development of nervous system,the maintenance of synaptic plasticity,the formation of neuronal circuits and the learning and memory processes The uptake and absorption of Glu in physiological state is a dynamic equilibrium process,Glu is mainly stored in the vesicles in the synaptic neurons,releasing Glu to the synaptic gap when the synaptic neurons are stimulated.The Glu in the synaptic gap binds to the glutamate receptor on the synaptic membrane and transmits further downward signals,thus giving play to the neurotransmitter function of Glu.At this time,the extra Glu in the synaptic gap will be ingested through the"Glu-Gln cycle"and stored in the synaptic vesicles again.When the nervous system is damaged,the Glu in the cell gap can’t be quickly ingested,resulting in the accumulation of extracellular Glu,which will lead to excessive activation of glutamate receptor and excitatory toxicity.NMDA receptor is an ionic glutamate receptor,which is related to learning and memory.Morris’s study found that the slow centripetal chamber injected with NMDA receptor blocker（AP5）can significantly inhibit the hippocampal synaptic transfer in rats with long potentiation（LTP）and spatial learning and memory ability.LTP is considered as the cell model for study and remember.Under normal physiological condition,the Glu in the synaptic gap activates the NMDA receptor and induces LTP.However,in pathological condition,the overexpression of NMDA receptor will cause the continuous development of ion channels,triggering a large number of Ca²⁺,resulting in neuronal death.Based on the above evidence,this study intends to explore the molecular biological mechanism of the rat learning and memory impairment induced by La.This study provides reliable experimental data and scientific basis for finding molecular biological targets of learning and memory impairment caused by lanthanum.Methods:Male and female Wistar rats（260±10g body weight）were purchased from the Experimental Animal Centre of China Medical University.All animals were housed in standard laboratory environment（temperature,22±1°C;humidity,55±5%）and observed for 1 week before mating.According to the principle of randomization,female and male rats were mated（male:female=1:1）.The presence of the copulatory plug indicated successful mating and the first day of pregnancy.During the period of pregnancy and lactation（6 weeks）,female rats were exposed to LaCl₃ in distilled drinking water at one of four doses:0%LaCl₃,0.125%LaCl₃,0.25%LaCl₃or 0.5%LaCl₃.And they were allowed to nurture their offspring normally.The offspring rats were exposed to LaCl₃ through both the placenta and lactation.After weaning,they received 0%,0.125%,0.25%or 0.5%LaCl₃ in drinking water for 4weeks.The animal models were used to simulate the children resided in high-REEs regions.So the exposure time of rats included pregnancy,lactation and one month after weaning（equal to 12 years old children）.The Morris Water Maze and the shuttle box test were used to determine the learning and memory ability of rats,and the electrophysiological test was used to observe the induction and maintenance of LTP in hippocampal CA1 region of rats,the ultrastructural changes of hippocampal neurons in rats were observed by transmission electron microscope,and the contents of Glu and Gln in extracellular fluid of rat hippocampus CA1 were detected by micro dialysis combined with high performance liquid chromatography.The real time PCR test and the Western blot test were used to determine the m RNA transcription and protein expression levels of GLAST、GLT-1、GS、PAG、GluN1、GluN2A、GluN2B in hippocampus and cortex of rats.In vitro experiment,the wistar pups were used to do the co-culture of primary neurons and astrocytes.The neuron-specific enolase（neuron-specific enolase,NSE）and glial fibrillary acidic protein（glial fibrillary acidic protein,GFAP）were used to identify the neurons and astrocytes.The micro dialysis and high performance liquid chromatography were used to detect the content of Glu and Gln in culture solution,and the real time PCR test method and Western blot method were used to detect the GLAST、GLT-1、GS、PAG、GluN1、GluN2A、GluN2B level of mRNA transcription and protein expression in cells.The TUNEL test was used to determine the neuron apoptosis/necrosis rate.After the intervention of MK801 and DCKA,the related indexes of neurons were measured again to verify the adverse effects of NMDA receptor induced by lanthanum-induced"Glu-Gln cycle"on astrocytes and neurons.Results:1.LaCl₃ exposure caused the accumulation of lanthanum in the hippocampus and cortex of rats.2.In the Morris water maze test,the escape latencies and the swimming distance of the rat increased with the increase of the dose of La.The control rats found the platform more quickly and directly and showed clear purpose,whereas the paths taken by the LaCl₃-treated rats were disorganized and purposeless.In the spatial probe trial,the time spent in the target quadrant and the number of target quadrant crossing of the LaCl₃-treated groups was significantly shorter than the control group.After removing the hidden platform,the control rats performed an explicit search strategy,frequently crossing the platform area in the target quadrant.However,the strategy of the LaCl₃-treated rats was purposeless,and these rats seldom went into the target quadrant,especially the 0.5%LaCl₃-treated rats.In the shuttle test,the number of electric shocks,the time of electric shock and the latency of active escape were significantly higher in the La group than in the control group,and the learning and memory ability damaged.3.In electrophysiological experiments,before high frequency stimulation,the PS average amplitude of each group was slightly decreased,but there was no difference among the four groups.After high frequency stimulation,the enhancement rate of PS in CA1 area of the hippocampus in La exposed group was significantly lower than the control group,and LTP was inhibited.4.The neuronal ultrastructure in the CA1 region of the hippocampus was altered with the increasing LaCl₃ doses.There were clear neuronal karyotheca,well-distributed chromatin and rich cytoplasmic organelles in the CA1 region of the control rats.LaCl₃-induced changes in the neuronal ultrastructure included indistinct nuclear membranes,changed neuronal karyotheca,invisible cytoplasmic organelles,swollen mitochondria and nuclear fragmentation.5.La exposure led to the increase of Glu in the CA1 area of the hippocampus and the cultured medium.6.In vivo and vitro test,results showed that LaCl₃ caused the down-regulation of GLAST,GLT-1,GS and PAG transcription and expression levels,and the up-regulation of GluN1,GluN2A and GluN2B.7.The effect of LaCl₃ on neuron apoptosis rate.In vitro,with the increase of LaCl₃ treatment concentration,the apoptosis rate of neuron cells significantly increased.8.The protective effect of MK801 and DCKA on neurons.The neurons were pretreated with MK801 and DCDA,which lowered the transcription and expression levels of NMDA receptor subunit GluN1,GluN2A and GluN2B,and decreased the neuronal apoptosis rate.Conclusion:1.LaCl₃ exposure caused the accumulation of lanthanum in the hippocampus and cortex of rats.2.La decreased the learning and memory ability of rats and the LTP.3.La increased the content of extracellular Glu,disturbed the"Glu-Gln cycle"cycle and over-activated the NMDA receptor.4.La damaged the neuronal ultrastructure and increased the neuron apoptosis rate.5.MK801 and DCDA pretreatment reduced the expression of NMDA receptor and the damage of lanthanum on nerve cells.