Study On Relationship Between Changed Function Of NMDA Receptor And Learning Memory Injury In Rats Caused By Arsenic

Background and purposeArsenic is a naturally occurring element widely present in the environment. Worldwide, more than 20 industrialized and less industrialized countries have drinking water contaminated with arsenic. There are more than 50 million people exposed to groundwater arsenic concentrations above the World Health Organization maximum permissible limits 50μg/L, many thousands of people developed arsenicalism. The three most affected areas in the world are Bangladesh, India and China. In both these areas, most of the source of arsenic is geological in origin, contaminating aquifers which provide water for over one million tube wells. In China, more than 5.6 million people are exposed to groundwater arsenic concentrations above 50μg/L, and more than 14.66 million people are exposed to groundwater arsenic concentrations above 10μg/L. Moreover, there are many people exposed to high levels of arsenic from burning coal.Chronic arsenic toxicity results in multisystem disease, such as peripheral neuritis, skin lesions (Hyperpigmentation, palmar and solar keratosis), peripheral vascular disease, hypertension, Blackfoot disease, and high risk of cancers. Young children (i.e., ages 0–6 years) may receive a much higher daily dose of chemical per-unit-body-weight than a lifetime average daily dose, particularly for chemicals in soil, because of their greater hand-to-mouth behavior than adults. Young children may also be more sensitive to the toxic effects of some chemicals than older age groups because of developmental and physiological differences. Epidemiological investigations found that arsenic toxicity can cause central nervous system impairment in adolescents, and the main manifestations of it are impairment of learning and memory. Animal experiments also found arsenic toxicity can impair the ability of learning and memory in rats. Up to now, the mechanism of the learning and memory impairment are poorly understood.The hippocampus is a part of the limbic system that is crucial to memory function and spatial navigation. Long-term potentiation (LTP) is widely accepted as a cellular and molecular model of information processing and storage by neural network. N-methyl-D-aspartate receptor (NMDAR) is one member of the glutamate-gated ion-channel receptor, with a high permeability to Ca2+ ions. To date, three main families of NMDAR subunits have been identified: NR1, NR2 (A-D) and NR3 (A-B). Many of the important NMDAR properties are influenced by the subunits composing the receptor assembly. NMDAR widely expresses in the central nervous system, and plays key roles in excitatory synaptic transmission. NMDAR subunit composition and its postsynaptic signaling pathway have important role in the induction and maintenance of LTP. Literatures have reported that acute and chronic arsenic exposure can significantly inhibit the induction and maintenance of LTP. So, we presume that arsenic exposure can impair the ability of learning and memory in adolescent through influencing the NMDAR subunit composition and its postsynaptic signaling pathway.The purpose of this study is to investigate the possible mechanism of arsenic neurotoxicity. We chose weaning Sprague-Dawley male rats as experimental model. The rats were assigned to four groups randomly (namely control, group A, group B and group C) and fed with water respectively containing 0, 2.72, 13.6 and 68 mg/L sodium arsenite for 1, 2, 3 months respectively. We will focus on the effects of arsenic exposure on learning and memory, the expression of NMDAR subunits and its postsynaptic signaling pathway.Methods1. A total of 180 weaning Sprague-Dawley male rats (40–50 g) were obtained from the Experimental Animal Center of this university, and kept in SPF laboratory animal room. The rats were assigned to four groups randomly and fed with water respectively containing 0, 2.72, 13.6 and 68 mg/L sodium arsenite for 1, 2, 3 months respectively. The As-containing water was freshly prepared every 2 days. Water used was free from bacterial contamination. Rats had free access to food.After arsenic exposure, samples of hippocampus were collected from two rats in each group for transmission electron microscopic study, and 10 rats from each group were tested in Morris water maze according to a modified procedure of Morris. At the end of spatial learning task, these rats were anesthetized with sodium pentobarbital by i.p. injection. Blood was collected by heart puncture; brains were separated and immediately transferred into liquid nitrogen, then stored at -70℃.2. The level of arsenic was determined using hydride generation atomic fluorescence spectrometry. Briefly, 0.2 g brain samples or 0.5ml blood were digested with 3 ml of concentrated HNO3 and 2 ml of concentrated H2O2 in microwave digestion system. Before measurement, added 10 ml 5% ascorbic acid and 5% sulfocarbamide to the digested samples then diluted to 50 ml with 5% hydrochloric acid. A blank digest was carried out in the same way. The concentration in diluted samples was determined in an atomic fluorescence spectrometer. The accuracy of the method was verified by standard reference materials.3. The acquisition of spatial learning and memory was assessed in the Morris water maze on three different components: hidden platform acquisition, probe trial and subsequent visible platform test.4. The hippocampus samples of 1 mm3 cube were dissected from CA1 area under anatomical microscope. After fixed in 2.5% glutaraldehyde, the specimens were sequentially processed with 1% osmium tetroxide, graded ethyl alcohols and embedded in EPON 618. The thin sections on copper mesh grids were stained with the heavy metals, uranyl acetate and lead citrate for contrast. After drying, the grids were then viewed on a transmission electron microscope.5. The expression of NMDAR subunits (NR1,NR2A,NR2B) in hippocampus were detected by the means of RT-PCR, Western-blot and immunohistochemistry. The hippocampi were collected from the rats exposed to arsenic for three months.6. To investigate the effect of arsenic exposure on the postsynaptic signaling pathway, we detected several important molecular. The activity of CaMKII was assayed with the SignaTECT Protein Kinase Assay Systems. Protein expression of CaMKIIα, p-CaMKIIα, PSD-95, SynGAP and ERK1/2 were detected by the means of Western-blot.Results1. General appearance and physical condition of arsenic-exposed and control rats were closely observed and no obvious difference was noticed. There were no differences in food intake between groups during the study. Compared with the rats in other three groups, rats in group C gained less body weights from the 6th week to the end of the study (P<0.05). Rats in group A and B did not show significant differences in body weight comparing with control rats. 2. Blood arsenic content increased with the concentrations of arsenic in drinking water, and there was significant difference among the treatment groups for three exposure time respectively (P<0.05). The arsenic content in brain increased with the concentration of arsenic in drinking water. The statistical significances were present between individual experiment group and control for three exposure time respectively (P<0.05). The results indicated that we have successfully raised the arsenic poisoning rats.3. There was no obvious impairment of spatial learning in rats exposed to arsenic for 1 and 2 months. After 3 months arsenic exposure, the rats in group C exhibited significant deficit in hidden platform acquisition compared with control and group A (P<0.05). Rats in group A and B did not show significant deficit comparing with control rats at the end of arsenic exposure. There were no significant difference in the spatial probe test and visible platform trial among the four groups. The results indicated the impairment of spatial memory in rats of group C.4. In the hippocampus of arsenic-exposed rat, individual shrunk cells could be seen with condensed cytoplasm and nucleus. The mitochondrion became swollen and vacuolized along with the cristae disorder and less in number. Rough endoplasmic reticulum presented sacculated distension. Vascular endothelial cells presented swollen mitochondria and the lumen of blood vessel irregular in the hippocampus of arsenic exposed rat. Edema around the capillary was obvious. The quantity of synaptic vesicle in synapse was decreased, and edema inside cells and organelles were confirmed. These ultra-structural changes became worse with increasing dose of arsenic exposure.5. A decreasing trend of NR2A mRNA and protein levels in hippocampus was observed after arsenic exposure. Decreases of NR2A mRNA and protein expression in hippocampus of rats treated with arsenic was in a dose-dependent manner (P<0.05). The expressions of NR1 and NR2B in hippocampus were not affected by arsenic toxicity.6. The activity of CaMKII in hippocampus was decreased significantly after arsenic exposure (P<0.05), while the protein expressions of CaMKIIαand p-CaMKIIαwere not affected. After arsenic exposure, the protein expressions of PSD-95, ERK1/2 decreased significantly (P<0.05). On the contrary, the protein level of SynGAP increased significantly (P<0.05). Conclusion1. After arsenic exposure from drinking water, arsenic can be rapidly absorbed into the blood circulation. Arsenic can penetrate the blood-brain barrier and accumulate in the brain. Its presence is associated with a series of ultra-structural pathological changes of the brain, and the impairment of spatial learning and behavioral tasks.2. Arsenic exposure can significantly inhibit the mRNA and protein expression of the NMDAR subunit NR2A. This change in NR2A was presented with the absence of changes in NR1and NR2B expression.3. Changed NMDAR subunit expression, lower CaMKII activity, lower expression of PSD-95 and ERK1/2 protein, together with higher expression of SynGAP protein after arsenic exposure, these results could inhibit the induction and maintenance of LTP. These changes maybe partly account for the molecular mechanism of learning and memory impairment caused by arsenic exposure.