Effects Of Lanthanum On The Learning, Memory And Ca²⁺/CaM-CaMKIV-CREB Pathway In The Hippocampus Of Rats

ObjectiveRare earth elements(REEs) include lanthanum(La),cerium,praseodymium, neodymium,promethium,samarium,europium,gadolinium,terbium,dysprosium, holmium,erbium,thulium,ytterbium,lutetium,scandium and yttrium in the periodic table of elements.In recent years,rare-earth techniques have been applicated widely in many fields such as agriculture,farming,aquaculture,industry and medicine in China. In particular,trace fertilizer and feedstuff containing REEs are used widespread in agriculture and farming.With widespread applications,more and more rare-earth REEs enter into the environment and accumulate in human body by food chain.Residue, accumulation of REEs in the environment and their effects on human health should not be ignored.Therefore,the effects of REEs on the environment and human health deserve further investigation.It is shown that REEs have hormesis effects.That means that low-level REEs can promote the growth of organisms,but high-level REEs have adverse effects on many tissues and organs such as brain,liver,kidney,heart and so on.Surveys on population have reported that average intelligence quotients,cognitive functions such as learning and memory of children living in the REEs-high regions were significantly lower than those of control children.Experimental studies have also shown REEs could significantly impair neurological behavior,learning and memory of animals.Nowadays, effects of REEs on central nervous system have been paid more attention.La is a light REE with active chemical properties,and it can react with almost all elements and components of tissues.Among REEs,La is also abundant in the environment. Furthermore,there is more accumulation of La in the brain.Therefore,La is often used as a representative to probe into the neurological adverse effects of REEs.Nowadays, mechanism underlying the learning and memory impairments induced by La is still not clear.Hippocampus is the key brain region related with learning and memory. Ca2+/CaM-CaMKIV-CREB pathway plays an important role in signal transduction associated with learning and memory.However,there is no reports that whether there are changes in this signal pathway induced by La or not.Animal model of learning and memory impairment was made in this study by subchronic administration of lanthanum chloride(LaCl₃) to rats from birth to weaning for one month.By neuroethological,biochemical,molecular biological and immunohistochemical methods and techniques,the effects of La on Ca2+/CaM-CaMKIV-CREB signal pathway were investigated,and the relationship between main components in signal pathway and changes in synaptic plasticity and neuroethological performances were discussed.The investigation of signal transduction mechanism underlying learning and memory impairments induced by La from molecular level could provide basic data and scientific reference for further elucidation of neurotoxic mechanism of La and even REEs.Based on these data,intelligence impairment and cognition deficiency caused by La and even REEs could also be prevented effectively.MethodsSixty Wistar rats weighing(260±10)g were obtained from the Center for Experimental Animals of China Medical University.The ratio of female to male is 2. Rats were housed at the standard laboratory condition with environmental temperature of(17～23)℃and humidity of 45～55%.The food was provided by Center for Experimental Animals of China Medical University.Animals were observed for 7 days before mating.Female rats were divided randomly into four groups:control,low-, middle- and high-dose LaCl₃ groups.Female and male rats were mated(female:male= 2:1).Occurrence of copulatory plug or sperm in the vaginal discharge in the second day after mating was used to indicate successful mating and day 0 of pregnancy.During the period of lactation(3 weeks),the control lactational rats drinked distilled water,and lactational rats in low-,middle- and high-dose LaCl₃ groups were exposed to LaCl₃ (99.9%,China) in distilled drinking water under one of three doses(0.25%,0.5%,and 1.0%).The pups were firstly exposed to LaCl₃ by parental lactation for postnatal 3 weeks and then orally administrated with 0.25%,0.5%,and 1.0%LaCl₃ in distilled drinking water for 1 month,respectively.La contents in the hippocampus of pups were determined by inductively coupled plasma mass spectrometry.Learning and memory of pups were measured by neuroethological tests including water maze,shuttle box and jumping stand tests.The expression of Nissl body in the hippocampus of pups was observed by Nissl staining.The ultra-structural features of neurons,synapses in the hippocampus of pups were observed with a transmission electron microscope.The contents of excitatory amino acid such as glutamic acid and aspartic acid in the hippocampus of pups were determined by o-phthalaldehyde precolumn derivatization with high performance liquid chromatography.The concentrations of intracellular calcium ions in the hippocampus of pups were detected by Fura-2 fluorescent probe. The activities of calmodulin(CAM) in the hippocampus of pups were determined by phosphodiesterase(PDE).The phosphorylated levels of calmodulin dependent protein kinaseâ…£(CaMKIV) and cAMP response element binding protein(CREB) in the hippocampus of pups were determined by western blot.The mRNA expression levels of c-fos and BDNF in the hippocampus of pups were determined by reverse transcription polymerase chain reaction(RT-PCR).The protein expression levels of c-Fos in the hippocampus of pups were measured by immunohistochemistry.Results1,Effects of LaCl₃ on learning,memory and ultra-structure in the hippocampus of ratsLa contents in the hippocampus of LaCl₃-administrated pups were significantly higher than those of control group.With LaCl₃-administrated dose increasing,La accumulation in the hippocampus of pups increased.In the water maze test,wrong number and latency of low- and middle-dose LaCl₃ groups were significantly higher than that of control group,and wrong number and latency of high-dose LaCl₃ groups were significantly increased as compared with than that of control and low-dose LaCl₃ groups.In the shuttle box test,shock number,time and latency of active avoidance in shock stimulation of middle-dose LaCl₃ groups were significantly higher than that of control group,and these parameters of high-dose LaCl₃ groups were significantly increased as compared with than that of control and low-dose LaCl₃ groups.In the jumping stand test,latency of low-dose LaCl₃ group was significantly lower than that of control group.As to middle-dose LaCl₃ group,wrong number was significantly increased as compared with than that of control group,but latency was significantly lower than that of control and low-dose LaCl₃ groups.As to high-dose LaCl₃ group, wrong number was significantly increased as compared with than that of control and low-dose LaCl₃ groups,but latency was significantly lower than that of control,low-and middle-dose LaCl₃ groups.The expression of Nissl body in CA1,CA3 and DG area of neurons in the hippocampus of LaCl₃ groups were lower significantly than those of control group.The expression of Nissl body in CA1,CA3 and DG area of neurons in the hippocampus of pups decreased further with administrated-dose increasing. Furthermore,Nissl body in CA1,CA3 and DG area of some neurons in the hippocampus of high-dose LaCl₃ group disappeared,and there was vacuolar degeneration. Besides,it was shown in this study that there were damages in the ultra-structures of neurons in the hippocampus of LaCl₃-administrated groups.Besides,there were ultra-structure changes in the synapses of hippocampus in LaCl₃-administrated pups. These ultra-structure changes included short active zone,small synaptic curvature and thin post-synaptic density.2,Effects of LaCl₃ on excitatory amino acids,Ca²⁺/CaM and CaMKIV in the hippocampus of ratsThe contents of glutamic acid and concentration of intracellular calcium ions in the hippocampus of low-dose LaCl₃ group were significantly higher than those of control group,but the activities of CaM and expression levels of p-CaMKIV were significantly decreased as compared with those of control group.The contents of glutamic acid and concentration of intracellular calcium ions in the hippocampus of middle-dose LaCl₃ group were significantly higher than those of control group,but the activities of CaM were significantly decreased as compared with those of control group, and the expression levels of p-CaMKâ…£were significantly lower than those of control and low-dose LaCl₃ groups.As to high-dose LaCl₃ group,the contents of glutamic acid and concentration of intracellular calcium ions in the hippocampus were significantly higher than those of control,low- and middle-dose LaCl₃ groups,and the contents of aspartic acid in the hippocampus were significantly increased as compared with control and low-dose LaCl₃ groups.However,the activities of CaM of high-dose LaCl₃ group were significantly lower than those of control and low-dose LaCl₃ groups,and the expression levels of p-CaMKIV were significantly lower than those of control,low-and middle-dose LaCl₃ groups.3,Effects of LaCl₃ on CREB,BDNF and c-fos expression in the hippocampus of ratsThe expression levels of p-CREB,c-fos mRNA and c-Fos in the hippocampus of low- and middle-dose LaCl₃ group were significantly lower than those of control group. The expression levels of p-CREB,c-fos mRNA of the hippocampus and c-Fos in DG area of the hippocampus in high-dose LaCl₃ group were significantly lower than those of control,low- and middle- dose LaCl₃ groups.The expression levels of c-Fos protein in CA1,CA3 area of the hippocampus in high-dose LaCl₃ group were significantly lower than those of control and low-dose LaCl₃ groups.As to the expression levels of BDNF mRNA of the hippocampus,there were no differences between control and low-dose LaCl₃ group.The expression levels of BDNF mRNA in the hippocampus of middle-,and high-dose LaCl₃ group were significantly lower than those of control and low-dose LaCl₃ groups.Conclusion1,Subchronic exposure to LaCl₃ could lead to learning and memory impairment and damages in ultra-structures of neurons and synapses in the hippocampus of rats.2,Subchronic exposure to LaCl₃ could induce change in the contents of excitatory amino acids and disturbance of calcium homeostasis,decrease the activities of CaM and the phosphorylated levels of CaMKIV in the hippocampus of rats.3,Subchronic exposure to LaCl₃ could cause lower levels of phosphorylated CREB,decrease the expression level of c-fos and BDNF in the hippocampus of rats.4,Subchronic exposure to LaCl₃ did harm to learning and memory of rats,which is possibly related to change in the contents of excitatory amino acids,disturbance of calcium homeostasis,decrease in the activities of CaM and consequent down-regulation of CaMKâ…£-CREB signal transduction pathway in the hippocampus of rats.