Effects Of Postnatal Methylmercury Exposure On Expression Of NMDA Receptor MRNA And Spatial Learning

Mercury is naturally occurring heavy metal and exists in a wide variety of physical and chemical states. Inorganic mercury could be converted through methylation to methylmercury (MeHg) by microorganisms. MeHg is accumulated and biomagnified throughout the food chain, reaching its most toxic concentration in the larger and long-lived fish and some marine mammals that are consumed by humans. The intoxication of MeHg, well known for Minamata disease, commonly results from ingestion of MgHg-contaminated food.MgHg is transported across the blood-brain barrier by amino acid carrier and accumulates in the brain. It also across the placenta and accumulates in fetal blood, brain and other tissues. Its preferential target is the central nervous system especially in the developing period. The detrimental effects include mental retardation, cerebral palsy, deafness, blindness, dysarthria, neurological and neuropsychological deficits. In many cases, fetus has overt symptoms while their mother seems normal. Accordingly, there are so many studies both clinical and experimental about prenatal exposure to MeHg, and it is well established that the brain of fetus is more affected by MeHg than that of adults. What should not be neglected is that infants would also be exposed to MeHg through consumption of human milk in the early postnatal weeks. The risk to the infant of the same level of exposure are predicted to be much high than adult because of the developmental status of the nervous system. Separating prenatal and postnatal neurodevelopmental consequences of MeHg exposure remains an important issue. High-dose MeHg exposures result in clear evidence of neurological disorders, while behavioral abnormality could be the only parameter to when moderate to low dose considered. We employed rats at different postnatal developmental periods and treated them with low concentration of methylmercury chloride (MMC) in 7 consecutive days. The impairment on their spatial learning ability due to MMC was observed by Morris water maze test.MeHg accumulates in astrocytes and inhibits their glutamate uptake, resulting in high extracellular glutamate, which activates the N-methyl-D-aspartate (NMDA) receptor. NMDA receptor plays an important role in various physiological processes during brain development and maturation. NMDA receptors are unique among glutamate receptors in terms of their voltage dependence and high permeability to calcium. High intracellular calcium causes ROS (reactive oxygen specie) production and activation of NOS (nitric oxygen synthase). This combination of properties is central to the role of NMDA receptors in forms of synaptic plasticity, such as long-term potentiation (LTP), which is involved with learning and memory and in the maintenance and selection of certain excitatory synapses formed during the early stages of development. NMDA receptors are comprised of an essential NR1 subunit coassembled with one or two types of the NR2 subunit (NR2A–D), additional NR3 subunits (NR3A–B) arranged within a tetrameric or pentameric heterooligomer. Consequently, it is obvious that the special functional properties of NMDA receptors are highly dependent on the NR2 subtypes present in the receptor, which is expressed temporally and spatially. Taken these facts together, it is likely assumed that in some degree MeHg exposure during different postnatal age impairs cognitive function and synaptic plasticity may due to altering NMDA receptor 2 subunits expression in the developing brain and changes in activities of anti-oxidative enzymes.To directly assess this assumption, we measured the expression of NMDA receptor 2 subunits mRNA in both hippocampus and cerebral cortex by transcript-polymerase chain reaction (RT-PCR) and activity of SOD, GSH-Px and NOS in order to evaluate the oxidative status in rats'brain.The whole study is composed of the following three parts:Part I Effect on Spatial Learning Ability of Different Postnatal MMC Exposure Objective: To investigate the effect of different postnatal MMC exposure on spatial learning ability. Methods: We treated rats with MMC (5mg/kg)as MMC-treated group and 0.86% sodium chloride solution (5ml/kg) as control group, orally for 7 consecutive days. Each group was separated by different postnatal period treatment (PND7,PND14,PND28,PND60)with 15 rats in each subgroup. 5 rats of each subgroup were raised normally after treatment and tested at PND69 by Morris water maze. Tissues of 6 rats of each subgroup at the third day after the final administration and the tissues of animals involved in water maze task were used for mercury determination by AFS (atomic fluorescence spectrometry). Results: Mercury concentration in both hippocampus and cerebral cortex is elevated significantly in MMC-treated group (p<0.01), while the level of mercury decreased dramatically in PND7 and PND14 MMC-treated rats when measured after water maze test, with only cortex of PND28 (p<0.01) and both hippocampus and cortex of PND60 (p<0.01) still much higher compared with that of control group. The latency of PND7 and PND14 MMC-treated rats in water maze is much longer than control rats (p<0.01); however rats of PND28 and PND60 performed as good as the control rats (p>0.05). Conclusion: Concentration of mercury in brain would decrease with time went by once without MeHg exposure. The first two weeks after birth is crucial to MeHg exposure, which would result in impairment of learning and memory ability for life long.Part II Changes in Activity of Enzymes after Different Postnatal MMC Exposure Objective: To analyze the changes in activity of anti-oxidative enzymes and nitric oxide synthase (NOS) after different postnatal MMC exposure and investigate its underlying relationship with learning and memory ability. Methods: Three rats from each subgroup three days after the final administration were sacrificed and their hippocampus and cerebral cortex tissue were used for detecting activity of SOD, GSH-Px and NOS by commercial kits. Results: In MMC-treated group, activity of SOD decreased significantly in both hippocampus and cortex of PND7 and PND14 rats(p<0.01), in cortex of PND28(p<0.01); activity of GSH-Px decreased in hippocampus of PND14 and PND60(p<0.01), and in cortex in all subgroups(p<0.01); differently, activity of NOS elevated dramatically in hippocampus of PND7, PND14 and PND28(p<0.01), in cortex in all subgroups(p<0.01). Conclusion: The activity of anti-oxidative enzymes declined after MMC exposure, which would attribute to oxidative injury in brain; the activity of NOS elevated indicated that there would be an increase production of NO, which would react with ROS resulting in further and more serious oxidative injury and it would activate NMDA receptor as messenger leading to excitatory neurotoxicity. Changes in cognitive ability were perhaps dependent on these results in some degree.Part III Expression of NMDA Receptors 2 Subunits mRNA after Different Postnatal MMC ExposureObjective: To investigate the effect of expression of NMDA receptors 2 subunits mRNA on spatial learning ability after different postnatal MMC exposure. Methods: Six rats of each subgroup were sacrificed by decapitation 3 days after the final administration. Expression of NMDA receptor 2A, 2B, 2C subunits mRNA in rat cerebral cortex and hippocampus were measured by semi-quantitative reverse transcript polymerase-chain reaction (RT-PCR), withβ-actin as standard. Results: In MMC-treated group, levels of NR2A mRNA decreased, especially in both hippocampus and cortex of PND14 rats (p<0.01); levels of NR2B mRNA were reduced in hippocampus of PND7, PND14 and PND28 rats (p<0.01), and in cortex of PND7 rats (p<0.05); an increase level of NR2C mRNA was observed in hippocampus of PND7 rats (p<0.01). Conclusion: The altering expression of NR2 subunits in the developing brain after MMC exposure during different postnatal period would make overactivation of NMDA receptor which is pivotal to LTP progress and cognitive function development.The main conclusions:1) We found that accumulation of methylmercury in brain is similar after different postnatal exposure, with concentration in hippocampus slight higher than that in cerebral cortex of PND14 rats; levels of mercury in brain would decline to almost normal level, but the impairment of cognitive function during the early age (especially around PND14) would last for life long.2) A decrease in activity of anti-oxidative enzymes in both hippocampus and cerebral after MMC exposure was observed, which indicated the oxidative injury due to MeHg; the increasing activity of NOS implied that there would be a increase in NO production, which leads to further oxidative injury and excitatory neurotoxicity.3) The spatial expression and distribution of NR2A, NR2B and NR2C has been disturbed by MMC exposure in different postnatal age, especially in the early days, which may attribute to learning and memory deficits in some degree.