The Role And Signal Transduction Pathway Of AMPA Receptor Trafficking In Aluminum-induced Impairment Of Hippocampal LTP In Rats

PartⅠ The damage of acute and subchronic aluminum exposure onhippocampal LTP and AMPA receptor trafficking in ratsAlpha-amino-3-hydroxy-5-methylisoxazole-4-proprionic acid (AMPA)-type glutamatereceptors (AMPAR) are multimeric proteins composed of GluR1, GluR2, GluR3, and GluR4subunits that mediate the majority of fast excitatory transmission in the central nervous system.The trafficking of GluR1and GluR2from intracellular pools to synaptic sites alters synapticstrength and has been recognized as a central mechanism of LTP. Since Al has been reportedaffecting LTP, may it be related to AMPAR? However, few studies on the effects of Al onAMPAR have been performed. Surprisingly, in a previous study, we observed decreasedexpression of glutamate receptors, including AMPARs, in the hippocampus after subchronic Alexposure in rats.On the basis of the previous study on the effects of Al on AMPAR and the possibleinvolvement of AMPAR in LTP, the present study was aimed to investigate the effects of acuteand subchronic treatment with different doses of aluminum-maltolate complex (Al (mal)3) onLTP and the total and membrane levels of the GluR1and GluR2AMPAR subunits in the rathippocampus. Here, a possible link between LTP dysfunction and AMPAR expression followingAl exposure was also explored.Chapter Ⅰ Effects of acute Al exposure on LTP in ratsObjective To explore the effects of acute Al exposure on LTP in rats. Methods Theexperiments were performed on24adult male rats (weight220-250g) that were kept underconstant temperature and humidity conditions with free access to food and water. The rats wererandomly divided into four groups (6rats/group): control group, low-dose group, medium-dosegroup and high-dose group. The rats received one dose of5μL of saline (control group) or a single dose of Al(mal)3(2.43,12.15or60.75μg Al) over5min via intracerebroventricular (i.c.v.)injection. fEPSP in CA1region were recorded by field potentiation technique in vivo. Results The basalfEPSP of60.75ug grops in preinjection and in postinjection were （109±4）%and（111±7）%respectively, no significant difference（P>0.05）. The fEPSP amplitudes of the control group were221%±10%,195%±30%and190%±27%at1,30and60min after HFS, respectively. Theaverage fEPSP amplitudes of the2.43μg Al group were164%±13%at30min and157±8%at60min after HFS, which represented a slight but statistically significant decrease compared to thecontrol group (P<0.05); these values dropped to165%±21%,155%±11%and140%±13%in the12.15μg Al group (P<0.05) and further decreased to149%±8%,139%±12%and110%±7%inthe60.75μg Al group (P<0.05). Notably, the suppression of LTP by Al was apparent between theAl and control groups (P<0.05), and further suppression was apparent with increasing Alconcentrations. At same time, Al(mal)3did not affect paired pulse facilitation（PPF）ratio（P>0.05）. Conclusion Acute Al treatment obviously suppressd the LTP in rat hippocampal CA1region in a dose-dependent manner in vivo, probably through postsynaptic mechanism but notpresynaptic transmitter release.Chapter Ⅱ Effects of acute Al exposure on AMPA receptors trafficking in ratsObjective To explore the effects of acute Al exposure on AMPA receptors trafficking inrats. Methods Animals and treatments were same with chapterⅠ. The expression of AMPARsubunit proteins (GluR1and GluR2) in both total and membrane-enriched extracts from the CA1area of rat hippocampus were detected by Western blot assay. Results None of the threedosages (2.43,12.15and60.75μg of Al) of Al(mal)3induced significant changes in GluR1(P>0.05) or GluR2(P>0.05) in the total extracts. However, significant decreases in GluR1(P<0.05) and GluR2(P<0.05) were found in the membrane-enriched extracts. With increasing Alconcentrations, the expressions of GluR1and GluR2proteins gradually decreased. Comparedwith the control group, the GluR1and GluR2levels of12.15μg Al group and the60.75μg Algroup were significantly decreased (P<0.05). Conclusion Acute Al treatment produceddose-dependent decreases of GluR1and GluR2in membrane extracts but not in total extracts,suggesting that the trafficking of AMPA receptor subunits from intracellular pools to synapticsites during HFS-induced LTP was suppressed.Chapter Ⅲ Effects of subchronic Al exposure on LTP in ratsObjective To explore the effects of subchronic Al exposure on LTP in rats. Methods Theexperiments were performed on24adult male rats that were kept under constant temperature and humidity conditions with free access to food and water. The rats were randomly divided into fourgroups (6rats/group): control group, low-dose group, medium-dose group and high-dose group.The rats received saline (control group) or Al(mal)3(0.41,0.82, or1.23mg/kg) viaintraperitoneal (i.p.) injection for8weeks. Hippocampal LTP in CA1region were recorded byfield potentiation technique in vivo. Results The fEPSP amplitudes of the control group were154%±15%and139%±12%at30and60min after HFS, respectively. The average fEPSPamplitudes of the0.41mg/kg Al group were153±21%and131±18%at the same time, whichrepresented no significant decrease compared with the control group (P>0.05); these valuesdropped to132%±20%and117%±7%in the0.82mg/kg Al group (P<0.05) and furtherdecreased to106%±40%and85%±10%in the1.23mg/kg Al group (P<0.05). Notably, thesuppression of LTP by Al was apparent between the Al and control groups (P<0.05), and furthersuppression was apparent with increasing Al concentrations. Conclusion Subchronic Altreatment obviously suppressd the LTP in rat hippocampal CA1region in a dose-dependentmanner in vivo.Chapter Ⅳ Effects of subchronic Al exposure on AMPA receptors trafficking in ratsObjective To explore the effects of subchronic Al exposure on AMPA receptors trafficking inrats. Methods Animals and treatments were same with chapterⅢ. The expression of AMPARsubunit proteins (GluR1and GluR2) in both total and membrane-enriched extracts from the CA1area of rat hippocampus were detected by Western blot assay. Results In contrast to the acuteexperiment, subchronic Al treatment caused dose-dependent decreases in GluR1and GluR2thatwere not restricted to the membrane extracts(P<0.05), but were present in the total extracts (P<0.05). Compared with the control group, the GluR-1level of the1.23mg/kg Al group and theGluR-2levels of the0.82mg/kg Al and1.23mg/kg Al groups decreased gradually in the totalextracts (P<0.05). Similarly, in the membrane-enriched extracts, the GluR-1and GluR-2levelsof the0.82mg/kg Al and1.23mg/kg Al groups also decreased gradually (P<0.05). ConclusionSubchronic Al treatment produced dose-dependent decreases of GluR1and GluR2both inmembrane extracts and in total extracts, suggesting that the trafficking of AMPA receptor and theexpression of AMPA receptor subunit proteins was suppressed.PartⅡ Study on involvement of signal transduction pathway of AMPAreceptor trafficking in aluminum-induced impairment of hippocampal LTP inratsOn the basis of the previous study of the possible involvement of AMPAR in the damage on hippocampal LTP by aluminum, the present study was aimed to investigate the signaltransduction pathway of AMPA receptor trafficking.ChapterⅠThe role of RAS in the damage on hippocampal LTP by aluminum in ratsSection1Effects of acute aluminum exposure on RAS activity in rats hippocampusObjective To explore the effects of acute Al exposure on RAS activity in rats hippocampus.Methods The experiments were performed on24adult male rats that were kept under constanttemperature and humidity conditions with free access to food and water. The rats were randomlydivided into four groups (6rats/group): control group, low-dose group, medium-dose group andhigh-dose group. The rats received one dose of5μL of saline (control group) or a single dose ofAl(mal)3(2.43,12.15or60.75μg Al) over5min via intracerebroventricular (i.c.v.) injection.Following electrophysiological recordings, the test of RAS activity were conducted by ELISA.Results With the increase of Al exposed dose, RAS activity decreased gradually. Compared withthe control group, the RAS activity of the medium-dose and high-dose group decreasedsignificantly (P<0.05). Conclusion Acute Al treatment obviously suppressd the RAS activity ofrat hippocampus during HFS-induced LTP was suppressed.Section2The antagonism of RAS activator EGF on suppression of hippocampal LTP byaluminumObjective To explore the antagonism of RAS activator EGF on hippocampal LTP suppressedby aluminum. Methods The experiments were performed on24adult male rats that wererandomly divided into four groups (6rats/group): control group, EGF group, EGF+Al groupand Al group. The rats received one dose of5μL of saline (control group),60ng EGF （EGFgroup）,12.15μg Al+60ng EGF (EGF+Al group),12.15μg Al (Al group) over5min viaintracerebroventricular (i.c.v.) injection. Hippocampal LTP in CA1region was recorded by fieldpotentiation technique in vivo. Results The fEPSP amplitudes of the control group were194%±25%,179%±22%and156%±27%at1,30and60min after HFS, respectively; Theaverage fEPSP amplitudes of the Al group were160%±4%,132%±15%and115%±12%at thesame time, which represented a statistical decrease compared with the control group (P<0.05);The average fEPSP amplitudes of the EGF group were224%±28%and189%±14%at1,20min after HFS, which represented a statistical increase compared with the control group (P<0.05), but on difference was found at30,40,60min after HFS(P>0.05);The average fEPSPamplitudes of the EGF+Al group were183%±5%and158%±15%at1,20min after HFS, which represented a statistical return compared with the Al group (P<0.05), but on difference wasfound at30,40,60min after HFS(P>0.05). Conclusion The RAS activator EGF couldantagonize the early suppression of hippocampal LTP by aluminum.Chapter Ⅱ The role of PI3K/PKB signal transduction pathway of AMPA receptortrafficking in the damage on hippocampal LTP by aluminum in ratsObjective To explore the role PI3K/PKB signal transduction pathway of AMPA receptortrafficking in the damage on hippocampal LTP by aluminum in rats. Methods Animals andtreatments were same with chapterⅠsection2. Following electrophysiological recordings, thephosphorylation of GluR1S831和S845and the PKB activity of rat hippocampus were detectedby Western blot assay and ELISA. Results Compared with the control group, thephosphorylation of GluR1S831和S845and the PKB activity of Al group significantlydecreased (P<0.05), but these values of EGF group statistically increased(P<0.05); Comparedwith the Al group, these values of EGF+Al group obviously return (P<0.05). Conclusion Thedepression of AMPA receptors phosphorylation mediated by PI3K/PKB signal transductionpathway may relate to the damage on hippocampal LTP by aluminum in rats.Chapter Ⅲ The role of MAPK/ERK signal transduction pathway of AMPA receptortrafficking in the damage on hippocampal LTP by aluminum in ratsObjective To explore the role MAPK/ERK signal transduction pathway of AMPA receptortrafficking in the damage on hippocampal LTP by aluminum in rats. Methods Animals andtreatments were same with chapterⅠsection2. Following electrophysiological recordings, ERKactivity of rat hippocampus was detected by ELISA. Results Compared with the control group,the ERK activity of Al group significantly decreased (P<0.05), but the value of EGF groupstatistically increased(P<0.05).; Compared with the Al group, the value of EGF+Al groupobviously return (P<0.05). Conclusion The MAPK/ERK signal transduction pathway may relateto the damage on hippocampal LTP by aluminum in rats, but the involvement of AMPA receptorsphosphorylation is not clear.Conclusions:Acute and subchronic aluminum exposure obviously and dose-dependentlysuppressed LTP in the rat hippocampal CA1region in vivo.The suppression on LTP induced by aluminum may be related to both trafficking and decreases in the expression of AMPA receptor subunit proteins.RAS→PI3K/PKB→GluR1S831和S845signal transduction pathway may relate tothe damage on hippocampal LTP by aluminum in rats.RAS→MAPK/ERK signal transduction pathway may relate to the damage onhippocampal LTP by aluminum in rats, but the involvement of AMPA receptorsphosphorylation is not clear.