| Effect of Maternal Chronic Aluminium Exposure onLTP, the Activity of PKC and the Content of Ng inthe Hippocampus in Vivo of their OffspringsObjectiveRecently, it has become clear that Aluminium(Al) has toxicity if much of thataccumulate in human's body. Evidence of epidemiology has shown that Al can impairneuron easily and cause deficiency of learning and memory. The nervous systemdysfunction induced by Al needs a long and chronic progress which concerns to everystage of intelligent development.The hippocampus is the most important encephalic region relative to function oflearning and memory. Hippocampal long-term potentiation (LTP) is NMDAreceptor-dependent persistent enhancement of efficacy in synaptic transmission, it isreputed that LTP represents the most intensively studied synaptic model and neuralbasis of learning and memory in the mammalian brain. So investigating the effect of Alexposure on LTP and the biochemical indicators relative to the LTP synapticmechanism will help to elucidate the mechanism of Al damaging the learning andmemory. Although Al has been reported to impair LTP following administration invivo and in vitro, the underlying mechanisms of Al action on LTP are still unknown.Protein kinase C(PKC) and neurogranin(Ng) which is a postsynaptic neuronalprotein,PKC substrate play especially prominent roles in induction and maintainanceof LTP. Meanwhile,The stage of pregnancy and lactation are very important periodduring intelligent development..The present study had observed the effect of Alexposure at low and high dose during pregnancy and lactation of maternal rats on theinduction and maintainance of hippocampus LTP and the activity of PKC and thecontent of Ng of their offsprings,hope to elucidate the mechanism of the effect of Alon LTP at this stage at the level of PKC and Ng. Materials and MethodsMaterials:1. Experimental animals60 Wistar rats(150-200g),♀:♂为3:12. Experimental reagentsAlCl3 solution, goat monoclonal anti-neurogranin antibody, rabbit anti-goatantibody conjugated with alkaline peroxidase, PepTag Assay for non-radioactivedetection of protein kinase C.3. Experimental instrumentsMorris water maze system, low temperature refrigeration centrifuge, ultravioletspectro photometer, automatic running gel imaging system, upright fluorescencemicroscope, deep freeze refrigerator, homeothermia freezing microtome, Motic ImagesAdvanced 3.2 image analysis system.Methods1. Groups45 female Wistar rats were divided randomly into 3 groups: group A, group B andgroup C which drank distilled water, 0.2%AlCl3 solution and 0.4%AlCl3 solutionrespectively from a month before pregnancy to their offsprings weaning(21 days old).The offsprings from all 3 groups(control group, 0.2%-Al and 0.4%-Al grouprespectively) were drank distilled water after weaning. The offsprings (3-4 months old)were used for study.2. Behavioral testingStep-down test was used for evaluating the effect of Al on behavior. Recording thetime of the first step down (latency) and the number of shock in 5 min (error number).3. Electrophysiological recordingsRats were anesthetized by intraperitoneal injection of 20%urethane(6.5ml/kg) andmounted in a stereotaxic apparatus. A concentric bipolar stimulating electrode wasplaced in the Schaffer lateral region (coordinates: 3.8 mm posterior to bregma, 3.8 mmright 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 pulsestimulus. When PS was steady, first the PS was recorded for 30 min, then highfrequency stimuli(HFS) were applied to induce LTP. Single test stimuli were deliveredonce every min to assess resulting changes in the PSs over time.4. The activity PKC assay in hippocampusThe PKC activity in hippocampus was detected by agarose-gel electrophoresis.5. Ng content assay in hippocampusWestern blot method was used to determine content of neurogranin.6. The concentration of brain and blood Al assayWeigh 0.1-0.5g brain tissue or take suction 0.2～0.5 ml whole blood, add 5～8 mlviolet acid, blank together. Heat at low temperature to dissolve totally, and continue toheat to almost dry. Add 0.2% nitric acid to dissolve the residue, and metered volume to50 ml in volumetric flask, the brain and blood Al concentrations were determined byatomic absorption plumbago.7. Statistical analysisAll data were analyzed by SPSS 13.0, P<0.05 was considered statisticallysignificant. Experimental values were indicated as the mean±SD.Results1. Ethology testThe behavioural dada showed: compared to the control group, the latency of thefirst step down reduced, the error number in 5 min increased in 0.2%- and 0.4%-groups. These differences of the latency and the error number in 5 min were significantas compared to the control group.2. LTP recordCompared to the control group, the enhancement rate of PS amplitude in 0.2%-and 0.4%- groups reduced evidently after HFS and this reduction was significant, butthe difference between the two groups was statistically non-significant.3. The PKC activity in the hippocampusThe PKC avtivity of plasmalemma,cytoplasm and ratio of membrane PKCactivity to that in cytosol were decresesd respectivelly in 0.2%- and 0.4%- groups as compared to controls. But the diffrence of these between 0.2％- and 0.4％- Al groupsare statistically non-significant.4. The content of Ng in the hippocampusCompared to control groups, the content of Ng (gray level of Ngexpression)reduced gradually in 0.2％-and 0.4％-Al groups. Although the content of0.4％-Al group was also decreased as compared to 0.2％-Al group, this decrease wasstatistically non-significant.5. Blood and brain aluminium concentrationBlood and brain Al concentration in 0.2％-and 0.4％-Al groups were higher thanthose of control groups. Both the changes were synchronous.DiscussionThe priod of from pregnancy to lactation is critical in intelligent development ofinfant. The investigating its mechanism is very important to protect the infant fromneurogenic disease induced by Al. In this study, the results showed that chronic Alexposure during pregnancy and lactation impair the induction and maintainance of LTPin hippocampus of their offspings and the degree of impairment was concerned in theexposure dose. The result of behavioral test also reflected this relationship. The presentresults also showed that The PKC avtivity of plasmalemma, cytoplasm and ratio ofmembrane PKC activity to that in cytosol were decresesd respectivelly in 0.2％-and0.4％-Al groups as compared to controls. Therefore, The regulation of B-50 andNMDA-recepter phosphorylation whith are necessary to induction and maintainance ofLTP was damaged..The present results also showed that chronic Al exposure during pregnancy andlactation could decrease the content of Ng in hippocampus in their offsprings. Wesuggest that the cause of decreased content of Ng may is that the number of dendriticspines was decresed, for overload AI caused karyopyknosis and loss of hippocampalneurons. The present record suggest that alterd Ng may contribute substantially toAl-exposed-associated learning and memory disorders related to hippocampaldysfunction.In a word, Al affects PKC and Ng by many means which play an important role inLTP. It's need to be further investigations. Conclusion1. Matemal chronic Al exposure impaired the behaviour of memory in theiroffsprings.2. Maternal chronic Al exposure impaired the induction and maintenance of LTPin vivo of their offsprings.3. One of the probable mechanisms was that PKC activity and Ng content werereduced by Al in hippocampus in their offsprings. |
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