Effects Of Chronic Aluminum Exposure From Prenatal To Postnatal On LTP And NNOS, NO, Ng In Hippocampus Of Young Rats

IntroductionAluminum is one of the most abundant element on earth. Neurotoxicity occurs with aluminum accumulation in human's body. Evidence of epidemiology has shown that Al can impair neuron and induce learning and memory deficiency. In experimentally induced dementia animal models, 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 Al exposure.The hippocampus is the most important encephalic region relative to function of learning and memory. Hippocampal long-term potentiation (LTP) is NMDA 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 maternal and postweaning is a very important period during brain development. There is 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 NO has not been studied so far. The aim of present study is to observe the effects of aluminum exposure from prenatal to postnatal at different dose on learning and memory, the induction and maintenace of LTP, the expression of nNOS and Ng and the intracellular NO concentration in hippocampus of young 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 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 whole gestation and suckling period. Their offspring were distributed into three experimental groups: one control group; two exposed groups (0.2%-Al group and 0.4%-Al group) where aluminum exposure ended at postnatal day 90. And the room temperature is 18℃～23℃, relative humidity is 45%～55%.2. 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.3. 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. 4. 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 form 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 bregma, 3.8 mm lateral to the midline, 3.8 mm under cortex). Glass micropipette filled with 3 mol/L KC1 were placed at the CA1 region of hippocampus (coordinates: 3.3 mm posterior to bregma; 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 stabilized 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.5. Expression of nNOS positive cellsThe nNOS 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 slice by Metamorph/Evolution/BX51 colour pathological figure analytical system.6. NO content in hippocampusThe content of NO were tested by nitrate reductase method . Dislodge the haippocampus into physiological saline (4℃) by 1:9 of volume ratio after electrophysiological recording. According to the instruction to do.7. Expression of Ng in hippocampusDislodge the haippocampus into cold lysate by 1:6～1:9 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 Ng in hippocampus.8. Morphology of structural featuresObserve the structural features of the young rats of aluminum exposure from prenatal to postnatal by electron microscopic.9. Statistical analysisExperimental 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 blood and brain aluminumThe aluminum concentration in blood and brain tissue were significantly higher than the control group (P<0.01) and increased with exposure dose. Futhermore, the difference between the two exposed group was statistically significant (P<0.05 or P<0.01).2. 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 group was statistically significant (P<0.05 or P<0.01).3. The comparison of average PS enhancement rate after HFSWith the increase of exposure dose, the PS magnitude reduced evidently after HFS. As compared to the control group ,the two exposed groups were significant reduced (P<0.01), but the difference between the two exposed group was statistically non-significant.4. The comparison of nNOS positive cells in hippocampusWith the exposure dose elevated, the value of optical density (Integrated OD Average, IA) reduced gradually in two exposed group. The expression of nNOS positive cells of the CA1 and CA3 region of hippocampus were significantly decreased in the Al³⁺ exposed groups (P<0.01). Futhermore, the difference between the two exposed group was statistically significant (P<0.01).5. The comparison of NO contents in hippocampusThe content of NO were significantly reduced in Al³⁺ exposed groups (P<0.01), and reduced with exposure dose. Furthermore, the difference between the two exposed group was statistically significant (P<0.01).6. The comparison of the expression of Ng in hippocampusCompared to the control group, the expression of Ng in hippocampus were significantly reduced in Al³⁺ exposed groups (P<0.05 or P<0.01), and reduced with exposure dose, Futhermore, the difference between the two exposed group was statistically significant (P<0.01).7. Morphology of ultrastructural features of hippocampusElectron microscopic examination showed the cytomembrane and cytoplasm were dissolved and nucleus were damaged. Mitochondria, Golgi apparatus and endoplasmic reticula were also damaged.DiscussionThe research on brain impairment in the maternal and postweaning period was an important stage relative to intelligent development of infant. Its mechanism is very important to protect the infant from neurogenic disease induced by Al. In this study, the results showed that chronic aluminum exposure from prenatal to postnatal 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 reflect this relationship. The present results also showed that chronic Al exposure at this stage decreased the the expression of nNOS positive cells and Ng and NO content in hippocampus.Some reports showed that Al interferes with glutamatergic neurotransmission, signal transduction pathways association of NMDA receptors and [Ca²⁺]_i through many ways. Hermenegildo found that a decrease in the cerebellar content of calmodulin and NOS 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.In Al-treated rats, the reduce of Glu and NMDA receptors lead to decrease of intracellular Ca²⁺ concentration and contents of calmodulin, then result in the decrease of the expression of nNOS positive cells and Ng and NO content in hippocampus.Aluminum also damaged mitochondria and DNA, then impaired the neuronal cells.In a word, Al affects the expression of nNOS and Ng and NO concent in hippocampus by many means which played important roles in the induction and maintainance of LTP. It's needed to be further investigations.Conclusion(1) Chronic Al exposure from prenatal to postnatal impaired the behaviour of the learning and memory in young rats.(2) Chronic Al exposure from prenatal to postnatal impaired the induction and maintenance of LTP in young rats.(3) One of the probable mechanisms was that Al inhibited the expression of nNOS and decreased the concentration of hippocampus intracellular NO.(4) One of the probable mechanisms was that Al reduced the expression of Ng in hippocampus.(5) One of the probable mechanisms was that Al impaired the hippocampus neuron.