Effects Of Maternal Chronic Aluminum Exposure On LTP And Intracellular Ca～(2+) Concentration And The Expression Of CaMKⅡ In Hippocampus On Their Offspring

IntroductionAluminum is one of the most abundant element on earth. It has neurotoxicity if much of that accumulate in human's body. Evidence of epidemiology has shown that AI can impair neuron easily and cause deficiency of learning and memory. In animal models of experimentally induced dementia it has been demonstrated that there are a reduced capacity of learning and memory and the pathological change which is similar with Alzheimer's disease after A1 exposure.The hippocampus were the most important encephalic region relative to function of learning and memory. Hippocampal long-term potentiation (LTP) is NMDA receptor-dependent persistent enhancement of efficiency in synaptic transmission, it is reputed that LTP represents the most intensively studied synaptic model and cellular basis of learning and memory in the mammalian brain. So investigating the effects of aluminum exposure on LTP and the biochemical indicators relative to the LTP synaptic mechanism will help to elucidate the mechanisms of aluminum damaging the learning and memory. Although A1 has been reported to impair LTP following administration in vivo and in vitro, the underlying mechanisms of Al action on LTP are still unknown.The stage of maternal is a very important period during development. The present study has observed the effect of maternal aluminum exposure at different dose on the induction and maintenance of LTP and the intracellular Ca²⁺ concentration and the expression of CaMKâ…¡of hippocampus on their offspring, hope to elucidate the synaptic mechanisms of the effects of A1 on LTP at this stage.Materials and methods1,Group and exposureThree groups of adult Wistar rats (150-200g) were exposed to aluminum through drinking 0ï¼…(distilled water), 0.2g/100ml and 0.4g/100ml aluminum chloride (AlCl₃) solution, respectively, for 30 days prior to mating and during the whole gestation and suckling period. Their offspring were distributed into three experimental groups: a controls group; two exposed groups (0.2ï¼…-Al group and 0.4ï¼…-Al group) where aluminum exposure ended at postnatal day 21. And the room temperature is 18～23℃, relative humidity is 45％～55ï¼….2,Electrophysiological recordingsRats were anesthetized by intraperitoneal injection of 20ï¼…urethane (6.5ml/kg, ip) and mounted in a stereotaxic apparatus. A concentric bipolar stimulating electrode was placed in the Schaffer lateral from CA3 region (coordinates: 3.8 mm posterior to bregma, 3.8 mm right lateral to the midline, 3.8 mm under cortex ) and a recording glass microelectrode (coordinates: 3.3 mm posterior to bregma, 1.5 mm right lateral to the midline)was lowered into the CA1 region until the population spike (PS) was induced by single pulse stimulus. When PS was steady, first the PS was recorded for 30 min, then high frequency stimulation (HFS) were applied to induce LTP. Single test stimuli were delivered once every min to assess resulting changes in the PS over time.3,Memorial behavior testStep-down test was used for evaluating the effect of A1 on behavior. Recording the time of the first step down (latencies) and the number of shock in 5 min (error number) of memory.4,Intracellular Ca²⁺ concentration in hippocampusIntracellular Ca²⁺ concentration in hippocampal cells was measured by the technique of Fura-2/AM calcium ions fluorescence indicator in SPF. The excitationwave is 340nm and the emission wave is 510nm.5,The expression of CaMKâ…¡in hippocampusDislodged the hippocampus into cold lysate by 1:6-1:9 of volume ratio. Pulverizedby transonic wave at 4℃and centrifuged in 12000 r·min^-1 1 hour, then separated thesupernatant. According to the technique of Western blot measured the expression ofCaMKâ…¡in hippocampus.6,Morphology of ultrastructural featuresObserved the ultrastructural features of the offspring of maternal aluminumexposure by electron microscope.7,Brain and blood Al determinationsWeighed 0.1-0.5g brain tissue or took suction 0.2～0.5 ml whole blood, added 5～8ml violet acid, blanked together. Heated at low temperature to dissolve totally, and thencontinue to heat to ahnost dry. Added 0.2% nitric acid to dissolve the residue, andmetered volume to 50ml in volumetric flask. The brain and blood aluminum concentra-tions were determined by atomic absorption plumbago.8,Statistical analysisExperimental values were indicated as the mean±SD. Interclass data were testedby ANOVA. The latencies and error number is not normal distribution so by rank sumtest.Results1,The comparison of average PS enhancement rate after HFSWith the increase of exposure dose, the enhancement rate of PS amplitude inexposure groups reduced evidently after HFS. The two exposed groups as compared tothe control group, this reduction was significantly (P<0.01), but the difference betweenthe two exposed groups was statistically non-significant. 2,The comparison of memorial behaviourThe behavioral data showed: the latencies of the step down was significantlydecreased, however, the error number of the step down was significantly increased inthe Al exposed groups (P<0.01) as compared to the control group, and also to thedifference between the two exposed groups.3,The comparison of intracellular calcium of hippocampal cellsThe hippocampus intracellular Ca²⁺ concentration was reduced in 0.2%-Al groupas well as 0.4%-Al group. However, this reduction in 0.2%-Al group was statisticallynon-significant (P>0.05),but was significantly decreased in 0.4%-Al exposed group(P<0.01). The difference between 0.2%-Al group and 0.4%-Al group wassignificantly (P<0.05).4,The expressionof CaMKâ…¡in hippocampusCompared to the control group, the expression of CaMKâ…¡in hippocampus weresignificantly reduced in 0.2%-Al group as well as 0.4%-Al group (P<0.01).5,Morphology of ultrastructural features of hippocampusElectron microscopic examination showed the cytomembrane and cytoplasm weredissolved and nucleus were damaged. Mitochondria, Golgi apparatus and endoplasmicreticula were also damaged.6,The comparison of blood and brain aluminumThe aluminum concentration in blood and brain tissue were significantly higherthan the control group (P<0.05 or P<0.01), and increased with exposure dose. Both thechanges were synchronous.DiscussionThe research on brain impairment in the maternal period was an important stagerelative to intelligent development of infant. To investigate its mechanism is veryimportant to protect the infant from neurogenic disease induced by Aluminum. In this study, the results showed that maternal chronic aluminum exposure impaired theinduction and maintainance of LTP in hippocampus, and the degree of impairment wasconcerned in the exposure dose. The result of memorial behavior test also reflected thisrelationship.The present results also showed that maternal chronic Al exposure could decreasethe intracellular Ca²⁺ concentration in hippocampus, which was dose-dependent. Somereports showed that Al maight interfere with [Ca²⁺]_i by block the VDCCs; inhibit theexpression of NMDA receptor in hippocampus; interfere IP₃ then affect the Ca²⁺inflowing and degrade the intracellular Ca²⁺ release from calcium reservoir pool.In one aspect that Al decreased intracellular Ca²⁺ concentration, another one is Alcompeted with Ca²⁺ to integrate with CaM, moreover, altered it's configuration theninterrupted the contribution of Ca²⁺/CaMKâ…¡, interfered with phosphorylation cours.At same time Al also affected NMDA receptor and AMPA receptor, consequentlyinhibited the expression of CaMKâ…¡.Aluminum also damaged mitochondria and DNA, then impaired the neuronalcells.In a word, Al affected Ca²⁺ concentration and the expression of CaMKâ…¡inhippocampus by many means which played an important role in LTP. It's needed to befurther investigations.Conclusion1. Maternal chronic Aluminum exposure impaired the memorial behavior in rats.2. Maternal chronic Aluminum exposure impaired the induction and maintenanceof LTP in rats.3. Maternal chronic Aluminum exposure impaired the hippocampus neuron in rats.4. One of the probable mechanisms of Al was that decreased the concentration ofhippocampus intracellular Ca²⁺ and inhibited the expression of CaMKâ…¡.