Effect Of Postweaning Chronic Alluminum Exposure On LTP And The Expression Of Intracellural CaN And PKA In Hippocampus Of Rats

IntroductionAluminum is one of the most abundant element on earth.Neurotoxicity occurs with aluminum accumulation in human's body.Evidence of epidemiology had shown that Al can impair neuron and induce learning and memory deficiency.In experimentally induced dementia of animal models,it had been demonstrated that there was a reduced capacity of learning and memory and the pathological change which was similar with Alzheimer's disease after Al exposure.The hippocampus is the most important encephalic region relative to function of learning and memory.Hippocampal long-term potentiation(LTP) is NMDA (N-Methyl-D-Aspartate) receptor-dependent persistent enhancement of efficacy in synaptic transmission,it is reputed that LTP represents the most intensively studied synaptic model and neural basis of learning and memory in the mammalian brain.So investigation of the effect of aluminum exposure on LTP and the biochemical indicators relative to the LTP synaptic mechanism will help to elucidate the mechanism of aluminum damaging the learning and memory.Although Al 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 postweaning is a very important period during brain development. There is a few reports of Al effects on learning and memory and LTP at this stage,so we should pay close attention to its potential and direct developmental toxicity. Furthermore,literature retrieval shows that the direct effect of Al on calcineurin(CaN) has not been studied so far.The aim of present study is to observe the effects of postweaning aluminum exposure at different dose on learning and memory,the induction and maintenace of LTP,the expression of CaN and cAMP-dependent protein kinase A(PKA) in hippocampus of rats,trying to elucidate the synaptic mechanisms of the effects of Al on LTP at this stage.Materials and methods1,Group and exposureHealthy adult Wistar rats(90～120g) were exposed to aluminum through drinking 0%(distilled water) or 0.2g/100ml(represented by 0.2%-Al) and 0.4g/100ml (represented by 0.4%-Al)aluminum chloride(AlCl₃) solution,respectively,during the postweaning period.Aluminum exposure lasted for 90 days.And the room temperature is 18℃～23℃,relative humidity is 45%～55%.2,Learning and memory behavioral testThe step-down test was used to monitor the learning and memory ability.Learning ability test:recording the escape latency and the number of errors in 5 min.24 hours later,memory ability test:recording the step-down latency and the number of errors in 5 min.3,Electrophysiological recordingsThe extracellular micropipette recording technique was used to recording electrophysiological change.Wistar rats were anaesthetized with 20%urethane(6.5 ml/kg,i.p) and their heads were fixed in a stereotaxis instrument.The scalp was cut to expose skull.Population spike(PS) was evoked by electrical stimulation of Schaffer Collateral pathway from the CA3 to the CA1 region of hippocampus,using a concentric bipolar stimulating electrode located at the area CA3 of Schaffer Collateral (coordinates:3.8 mm posterior to bregrna,3.8 mm lateral to the midline,3.8 mm under cortex).Glass micropipette filled with 3 mol/L KCl was placed at the CA1 region of hippocampus(coordinates:3.3 mm posterior to bregrna;1.5 mm lateral to the midline; on the surface of cortex) and lowered into the CA1 region for recording field potential. After the response had stabililized in each rat,30 min baseline was recorded.Stimuli were given every minute(all responses were averaged for each date point).A high frequency stimulation(HFS:100 Hz,5 s) was delivered at the same intensity.Testing with single pulse was continued for 45 min after the HFS was delieved.4,Expression of CaN in hippocampusDislodge the hippocampus into cold lysate by 1:3 of volume ratio after electrophysiological recording.Pulverized by transonic wave at 4℃and centrifuged in 12000 r·min^-1 1 hour,then separate the supernatant.According to the technique of Western blot measured the expression of CaN in hippocampus.The CaN positive cells were observed with immunohistochemistry.The rats were fixed by heart perfusion with 4%paraformaldehyde after electrophysiological recording.Manufacture routine paraffin section.Analyze the slices by Metamorph/ Evolution/BX51 colour pathological figure analytical system.5,Expression of PKA in hippocampusDislodge the hippocampus into cold lysate by 1:3 of volume ratio after electrophysiological recording.Pulverized by transonic wave at 4℃and centrifuged in 12000 r·min^-1 1 hour,then separate the supernatant.According to the technique of Western blot measured the expression of PKA in hippocampus.6,Brain and blood Al determinationsWeighed 0.1～0.5 g brain tissue or took suction 0.2～0.5 ml whole blood,added 5～8 ml violet acid,blanked together.Heated at low temperature to dissolve totally,and then continue to heat to almost dry.Added 0.2%nitric acid to dissolve the residue,and metered volume to 50 ml in volumetric flask.The brain and blood aluminum concentrations were determined by atomic absorption plumbago.7,Statistical analysis Experimental values were indicated as the mean±SD.Interclass data were tested by ANOVA.The results of behavioral test are not normal distribution so by rank sum test.Results1,The comparison of learning and memory behavioural testThe behavioural data showed:the escape latency was remarkably prolonged (P＜0.01),the step-down latency was remarkably decreased(P＜0.01),and the number of errors both in learning and memory were significantly increased in the Al³⁺ exposed groups(P＜0.01) as compared to the control group.Futhermore,the difference between the two exposed groups was statistically significant(P＜0.05 or P＜0.01).2.The comparison of average PS enhancement rate after HFSWith the increase of Al³⁺ exposure dose,the PS magnitude reduced evideutly after HFS.As compared to the control group,0.4%-Al group was significant reduced (P＜0.05),but the difference between 0.2%-Al group and the control group was statistically non-significant.3.The comparison of the expression of CaN in hippocampusCompared to the control group,the expression of CaN in hippocampus was significantly increased in two Al³⁺ exposed groups(P＜0.01),and increased with exposure dose,Furthermore,the difference between the two exposed groups was statistically significant(P＜0.01).4.The comparison of the expression of PKA in hippocampusCompared to the control group,the expression of PKA in hippocampus was significantly reduced in two Al³⁺ exposed groups(P＜0.01),and reduced with exposure dose,Furthermore,the difference between the two exposed groups was statistically significant(P＜0.01).5.The comparison of blood and brain aluminum The aluminum concentration in blood and brain tissue increased with exposure dose;Furthermore,the difference in the aluminum concentration in brain tissue between the two exposed groups was statistically significant(P＜0.01).DiscussionThe postweaning brain impairment was an important stage relative to intelligent development of children.Its mechanism is very important to protect the children from neurogenic diseases induced by Al³⁺.In this study,the results showed that postweaning chronic aluminum exposure impaired the learning and memory ability,and the degree of impairment was concerned with the exposure dose.The results of induction and maintainance of LTP in hippocampus also reflected this relationship.The present results also showed that chronic Al exposure at this stage decreased the the expression of CaN and PKA in hippocampus.Some reports showed that Al interferes with glutamatergic neurotransmission, signal transduction pathways association of NMDA receptors through many ways. Hermenegildo found that a decrease in the cerebellar content of calmodulin in Al-treated rats.Al appeared to functionally alter the blood-brain barrier and produce changes in cholinergic and noradrenergic neurotransmission.Therefore,Al can interfere with more steps in signal transduction pathways and impair a series of targets during the induction and maintainance of LTP.Aluminum increased the expression of CaN and decreased the expression of PKA in hippocampus.In Al-treated rats,the reduce of intracellular Ca²⁺ concentration and contents of calmodulin lead to decrease of the activity of PKA.In a word,Al affects the expression of CaN and PKA in hippocampus by many means which played important roles in the induction and maintainance of LTP.It's needed to be further investigations.Conclusion1,Postweanining chronic Al exposure impaired the behaviour of the learning and memory in rats. 2,Postweanining chronic Al exposure impaired the induction and maintenance of LTP in rats.3,One of the probable mechanisms might be that Al increased the expression of CaN in hippocampus.4,One of the probable mechanisms might be that Al reduced the expression of PKA in hippocampus.