Changes Of Hippocampal NMDAR Subunit,[Ca～(2+)]_i,Apoptosis Induced By Acute Or Chronic Lead Exposure And Relationship With Impairment Of Learning And Memory In Pups Of Mice

ObjectiveThe heavy metal lead(Pb²⁺) is a well - known environmental neurotoxins. Due to nervous system being in stage of rapid development and incompletely developed blood brain barrier, children's nervous system were susceptive to lead and lead poisoning of children was more familiar and severe. Impairment of nervous system exposed to Pb²⁺ during development (pregnancy and lactation) maintained to adult. Blood lead level of infant and pre - school children was markedly correlated to IQ, higher blood lead level, less intelligence quotient. Even a low level of Pb²⁺ exposure ( blood concentration of Pb²⁺ as low as 100μg/L) can result in impairment of learning and memory and cognitive functions.Hippocampus was one of the key limbic regions involved in learning and memory, and LTP in hippocampus was one of mechanism of forming of memory. NMDA receptor exerted an important role on synaptic plasticity and related to learning and memory. In a subsequent paper, the author showed that we firstly set up a protocol for chronic exposure to lead, and then using a water maze paradigm and jumping stand test we measured the ability of spatial learning and Obtaining or maintaining ability of passive - avoidance reaction. In the present study, we investigated the expression of NMDA receptor subunit ε1,ε2 messenger RNAs(mRNAs) and proteins.During induction of hippocampal LTP, calcium ion took a key role. Firstly, Ca²⁺ influx into presynaptic membrane resulted in release of glutaminic acid and depolarizing postsynaptic membrane; Secondly depolarizing postsynaptic membrane and glutaminic acid bonding to NMDA receptor expelled Mg²⁺ Which blocked NMDA receptor channels, this lead to open NMDA receptor channels and Ca²⁺ influx into postsynaptic membrane, and lastly Ca²⁺ triggered LTP. Onthe other hand, intracellular Ca²⁺ overloading was one of causes leading to the death of nerve cells.AP - 5 was one of specific antagonists of NMDA receptor. Inhibited effect of lead on NMDA receptor took an precedent part to impairment of learning. Our past studies suggested that chronic exposure to Pb²⁺ could cause the increase of intracellular free Ca²⁺ concentration in hippocampal cells in rats after LTP. Studies showed that Pb²⁺ lead to imbalance of homeostasis of intracellular Ca²⁺ , but its mechanism and relationship with NMDA receptor channels and calcium store were incompletely clear. The present study investigated the mechanism of changes of intracellular free Ca²⁺ concentration in hippocampus induced by lead (Pb²⁺) using AP - 5 , heparin and procaine as tool drugs.The neurotoxicity of lead was related to apoptosis of brain neural cells. Now majority of studies indicated that apoptosis were mediated by transcript factor such as bcl - 2 family and p53 gene and so on, but minority of studies dealt with enzymes of apoptosis like Calpain and Calcineurin. Activation of Calpain and Calcineurin were dependent to intracellular free Ca²⁺ and thus induced degradation of structure protein. Our previous study indicated that AP - 5 reduced damages of hippocampal cells, and its mechanism was correlated to inhibit NMDA receptor channels. Was the protection of AP - 5 on hippocampal damages induced by lead correlated to apoptosis? The subsequent paper investigated that apoptosis resulted from lead exposure and relationship with activities of Calpain and Calcineurin in hippocampal cells of mouses. Futher to study and discovery the mystery of lead poisoning.Materials and MethodsThe protocols for chronic exposure to lead was as follow: Kunming mouses were purchased from animal department of china medical university and were individually housed in plastic cages and one male mouse was mated with two female mouses of same strain. Mouses were randomly and equally divided into four groups. On postnatal day 1, the dam began to be exposed to lead at 0, 4. 8, 9. 6, 14.4mmol. L^-1 in drinking water. Since weaning at postnatal 21d, the pups were directly provided lead in drinking water. Food and water were free providedto all animals. At postnatal 7d, 14d, 21d, 28d, individually the hippocampus were got from pups in different groups and stored at fluid nitrogen and then stored at -70℃ refrigerator. Since postnatal 28d the pups began to train in a spatial learning task using a water maze paradigm and in a passive escape reaction task using jumping paradigm. The expression of NMDA receptor subunit ε1,ε2 were determined by western blots and the expression of NMDA receptor subunit ε1,ε2,bcl -2 and bax gene were determined by RT - PCR.The protocols for acute exposure to lead were as follows: in order to acute lead exposure in vitro, the mouse brain was quickly removed and immersed in ice - cold ACSF bubbled with 95% O₂ + 5% CO₂, and 350μm hippocampal slices were prepared. After 2h stabilization, using acute hippocampal cells dis-sactated technique and Fura - 2 determined the concentration of free calcicum i-ons in hippocampal cells. The different concentrate lead acetate and other factors were added into the media, the slices was collected at different time. The control group of acute or chronic using water instead of lead acetate. The activ-ies of calpain and calcineurins were observed.Results1. Chronic lead exposure impairs spatial and passive - avoidance learning and memory in pups . of mouse.In the Morris water maze task, results indicated that mouses having drink lead water displayed significant impairment in their performance, and this extent of impairment showed in lead concentration dependent manner. In the jumping stand test, results suggested that lead exposure significantly reduced ability of acquiring and maintaining passive - avoidance reaction and this was correlated with concentration of drinking lead. 2. Expression of NMDA receptor subunit ε1- NR2A gene was reduced in hippocampus of pup mouses exposed to Pb²⁺ during development.Expression of NMDA receptor subunit ε1 - NR2A,ε2 - NR2B gene in hippocampus of pup mouses chronic exposured to lead were determined by using RT- PCR. The results indicate that decreased expression of ε1 - NR2A gene was found in hippocampus after Pb²⁺ treatment in dose - dependent manner. Statisti-cally, no change in the expression of e2 - NR2B gene in the hippocampus was apparent between control group and Pb²⁺ treated groups during development.3. Different trends in modulation of NMDAR ε1 - NR2A,ε2 - NR2B protein expression in the hippocampus of mouse after chronic lead exposure during development.Western blot analysis of the NMDA receptor subunits ε1 - NR2A,ε2 -NR2B in the hippocampus of mouses aged postnatal 7, 14, 21, 28 days after in vivo exposure to Pb²⁺ acetate. Mouse pups exposed to Pb²⁺ during development did express lower levels of ε1 - NR2A protein expression in the hippocampus than that of the control group and this exhibited in a dose - dependent and developmental expressed differences manner. No change in the expression of ε2 -NR2B subunit protein was observed in the hippocampus in mouses at any age exposed to Pb²⁺ during development.4. Effect of acute lead exposure on the concentration of free intracellular Ca²⁺ in hippocampal cells of mouses.Compared with control group, the levels of free intracellular Ca²⁺ in hippocampal cells were significantly increased in exposed to lead groups(P <0. 05) . This effect of Pb²⁺ exhibited time - dependence and dose - dependence.5. Effect of levels of extracellular calcium ions on changes of free intracellular Ca²⁺ induced by Pb²⁺Compared with control group, when levels of extracellular calcium ions was zero, lead exposure could significantly increase the levels of free intracellular Ca²⁺ in hippocampal cells, and this exhibited in a dose - dependent manner.6. Effect of Pb²⁺ on the increases of free intracellular Ca²⁺ evoked by gluta-mateLevel of 0.1 10μmol. L^-1 inhibited the increase of free intracellular Ca²⁺ in hippocampal cells evoked by glutamate.7. The protection of AP - 5 to damage induced by lead or glutamate and effect of AP - 5 on the inhibition of Pb²⁺ to increase of free intracellular Ca²⁺ induced by glutamate.Results suggested that AP - 5 could enhance the inhibition of Pb²⁺ to increases of free intracellular Ca²⁺ in hippocampal cells induced by glutmate, andinhibited rate was elevated from 78% in 10μmol. L^-1 Pb group to 93% in 50μmol. L^-1 AP -5 group. AP -5 exhibited the protection for impairment of hippocampal slice induced by Pb²⁺ or glutamate.8. Chronic expose to lead acetate affects Ca²⁺ release from different calcium stores.To study the effect of Pb²⁺ on the IP₃ - sensitive and ryanodine - sensitive calcium store, By using specific antagonist of IP₃ and ryanodine - heparin and procaine pretreatment respectively, the hippocampal cells were stimulated by lead acetate and its effects on the intracelluiar free calcium ions were analyzed. The levels of intracelluiar free calcium ions in pretreated by heparin or procaine decreased significantly compared with control. The results suggested lead acetate could activate free Ca²⁺ release from IP₃ - sensitive and ryanodine - sensitive calcium store.9. Acute lead exposure alters the activities of Calpain and Calcineurin and role of AP - 5The results indicated that the activities of Calpain and Calcineurin markedly increased in hippocampal slice, but AP-5 could block the effect.10. Effect of lead acetate on the expression of bcl - 2 and bax genes in hippocampus of mouse pupsThe expression of bcl - 2 was significantly decreased, whereas bax was sig-nigicantly increased in hippocampus in lead acetate treatment group compared with the control group.DiscussionNow there are two kind of models used in learning and memory: one is passive - avoidance learning, for example, jumping stand test, climbing mast and shuttle box, the other is discrimination learning, such as kinds of mazes. Jumping stand test mainly manifested passive - avoidance learning ability of pups. The latent period of escape in the first and the second stage of test were prolonged , whereas the latent period of stay in the same stage were shortened significantly respectively in lead acetate treatment group compared with the control group. The results suggested that chronic lead acetate expose reduced ability ofpassive - avoidance learning of mouse pups including acquiring and maintaining ability of passive - avoidance reaction. In the place navigation version of the Morris water maze task, mouse pups having lead acetate water displayed significant impairment in spatial learning and memory and lower ability of spatially locate and move.NMDA receptor take a double role in synaptic excitability transmitting, synaptic plasticity, learning and neuroexitotoxicity: on the on hand it intervenes with excitable signals transfers over membrane, on the other hand it results in death of cells glutamate free calcium ion entry into neurons by means of gluta-mate in stroke, brain injury and nerves degeneration. NMDA receptor was composed of ζ1 (means NR1 in rats) and el -4(means NR2A - D in rats) , its regular conformed pattern in ζ1/ε (means NR1/NR2 in rats), ε1 and ε2 protein mainly express in hippocampus and cortex. The present study showed that decreased trend of ε1 protein expression was found in hippocampus of mouse pus exposed to chronic lead acetate, but no changes of ε2 protein expression compared with the control.Lead could alter the function of NMDA receptor by different pathway: pre-treating with lead could inhibit NMDA receptor channels, and developmental brain tissue was more sensitive to lead exposure, therefore this suggested the effect of lead chiefly expressed in a specific developing period. Hippocampal NMDA receptor mRNAs underwent subunits specific changes during developmental lead exposure. Reduced NMDA receptor subuint NR1 and NR2A were found in hippocampus of Pb²⁺ - exposed mouses, but Pb²⁺ exposure during development had no effect on NR2B subunit mRNA in hippocampus at any age. Age - dependent and spatial - dependent changes in NMDAR subunit gene expression were observed in hippocampus of mouse chronically exposed to Pb during development. The early developmental NMDAR subunit protein was more sensitive to lead exposure than that of the lately. These finding concurred with our present results.Recently studies reported that higher expression of el gene binding ζ1 gene or el gene in hippocampus and cortex, stronger spatial learning and memory a-bility. Toscano suggested that deficits of learning and memory in Pb²⁺ - exposedrats during development was because of composition of NR1/NR2B of subunits comprising NMDAR being increased and to inhibit subunit NR2B transform into NR2A and further interfere to CREB phosphorylation dealing with Ca²⁺ - sensitive signal transduction. These findings indicated that the effect of lead exposure on expression of NMDAR subunit gene and protein was a key role for deficits of learning and memory.The current study showed that acute chronic lead exposure could result in dose - dependent and time - dependent increases of free intracellular Ca²⁺ in hippocampal cells and this change was not influenced by extracellular calcium ion. The finding indicated that the increases of free intracellular Ca²⁺ induced by lead was related to Ca²⁺ release from calcium stores. Further study provided evidence that acute lead exposure could activate Ca²⁺ release from IP₃ - sensitive and ryanodine - sensitive calcium stores.The increases of free intracellular Ca²⁺ in excitability cell can be triggered by depolarization of membrane and Ca²⁺ channel opening. The present study showed that lead could inhibit the increases of free intracellular Ca²⁺ induced by glutamate in dose - dependent manner and AP - 5 enhanced this inhibiting effect of lead and AP - 5 exhibited protection to damages of hippocampal slice evoked by lead and glutamate. These finding demonstrated that lead inhibited the opening of NMDA receptor channel induced by glutamate and the protection of AP -5 to damages of hippocampal slice evoked by glutamate or lead was dependent to block NMDA receptor channels. We presumed: Pb²⁺ and Ca²⁺ entered competitively cell by NMDAR channels, because intracellular free Pb²⁺ took a pseudo -calcium role and subsequently instead of free intracellular Ca²⁺ activated IP₃ and ryanodine calcium channel of Endoplasmic reticulum and releasing Ca²⁺ from a-bove calcium stores, whereas overload of intracellular Ca²⁺ resulted in death of nerve cells. When the NMDA receptor channels were blocked by AP - 5, the pseudo - calcium effect of Pb²⁺ was weaken and its neurotoxicity disappeared.The apoptosis in brain cells, especially in hippocampus, inevitably caused injury of center and limbic nervous system and then affect learning and memory. Now we still realizes clearly the mechanism of the apoptosis. Previous studies provided evidence that bcl - 2 family took part in cerebral ischemia and hypoxiainjury, Alzheimer disease and apoptosis of nerve cell induced by nitric oxide. The present study demonstrated that lead could induced apoptosis in mice hippocampus through the down regulation of bcl - 2 and the up regulation of bax gene expression, and there was a nice dose - response relationship.The mechanism of apoptosis induced by lead was unclear. According to our study, we considered as following factors: Firstly, disturbance of Ca²⁺ induced by lead. In vitro mice cell culture, Obert had observed that calcium antagonist could attenuated the apoptosis of granular cell in cerebellum evoked by lead. These finding indicated that Ca²⁺ played an necessary role in apoptosis resulting from lead. Ca²⁺ induced apoptosis of nerve cell by means of following passway: on the one hand, Ca²⁺ activated not only Ca²⁺ - dependent Calpain and Calci-neurin and then changed structure of chromosomes, but also activated endogenous nuclease and subsequently produced DAN ladder and apoptosis. In addition , many proteins including tubulin - 2 and nerve fiber protein were sensitive to Calpain. Activated Calpain lead to the lesion of cellular skeleton and plasma membrane, and lastly resulted in the death of neurons.Injury of mitochondrial induced by calcium overload made start factor of apoptosis releasing, such as cytochrome C, caspase activator and apoptosis induce factor. These could rapidly activated endogenous nuclease and caspase and lead to apoptosis. The current study indicated that activities of Calpain and Calci-neurin markedly increased in hippocampal slice exposed lead, but AP - 5 could block the effect. Secondly, disorder of NMDAR induced by lead. The present study and study of Guilarte suggested that chronic lead exposure altered the composition of NMDA receptor subunit, and suppressed the function of NMDAR。 Ikonomidou suggested in Science that the apoptosis and necrosis in brain cells at rats during development was induced after the NMDA receptor channel was blocked only a few hours. These finding suggest that the apoptosis evoked by lead may be related to the inhibition of NMDA receptor channels. Lastly, the previous study of our lab demonstrated that the expression of c - Fos gene and c - Jun gene of hippocampus exposed lead was elevated compared with the control. As transcript factor, c - Fos gene and c - Jun gene promoting apoptosis could correlated with regulation of associated apoptosis gene, therefore resulted...