The Effects Of Methamphetamine On Learning And Memory And Its Relationship With VGLUT1

Objective and significance:Methamphetamine(MA) is a widely abused central stimulant. In China, the illegal use of MA is now more serious than traditional narcotics such as heroin and cannabis. MA-induced neurotoxicity can lead to cognitive dysfunction, increasing the possibility of psychiosis, abnormality in emotions and behaviors. Cognitive dysfunction may manifest as impairment in learning and memory, as well as distinguish and control ability. On the other hand, low dose of MA was once used as a nootropic. Therefore, the exact effects of MA on learning and memory and the underlying mechanisms are still not well understood. There is also no effective therapy for the prevention and treatment of the dysfunction.Recently, increasing evidence revealed that glutamatergic transmission anomalies are associated with MA-induced learning and memory dysfunction. Glutamate transporters play essential roles in glutamatergic signal transmission. There are two types of glutamate transporters, excitatory amino acid transporters(EAATs) and vesicular glutamate transporters(VGLUTs). VGLUTs selectively transport glutamate in the cytoplasm into vesicles, and therefore control the concentration of glutamate in synaptic vesicles. Thus, the expresison and activity of VGLUT1 can determine the glutamate concentration in synaptic cleft, then the efficacy of synaptic transmission. There are three isoforms of VGLUTs, denoted VGLUT1, VGLUT2, and VGLUT3. VGLUT1 mainly distributed in the cortex and hippocampus which are associated with learning and memory. We have found that VGLUT1 expression is related with neural injury induced by MA, and VGLUTs inhibitor, CSB6 B, significantly attenuating neurotoxicity caused by MA and improving learning and memory impairment. But the dynamic influences of MA on learning and memory and how the VGLUT1 participate in this process is still unknown. Whether changing the anomalies concentrationof glutamate between the synaptic cleft through regulating expression and activity of VGLUT1 can influence the alteration of learning and memory caused by MA is not clear..In this study, we evaluate the effects of MA on different types of learning and memory. Taking some pathological observation,like neural injury in hippocampus and cortex,to evidence the impairment of learning and memory. Assaying the expression of VGLUL1 at behavior impairment time, compared with the nonimpaired time to clear the relationship between VGLUT1 and the behavior alteration induced by MA.This study would contributate to reveal the mechanism of VGLUT1 on learning and memory dysfunction induced by MA. Method:1. The influence of MA on learning and memory: Animals were tested on step-down test, Y-maze, Morris water maze and conditioned place preference models to evaluate the effect of MA on avoidence memory, identification memory and rewarding memory. Drugs are single injection and long-term exposure.2. The influence of MA on general morphology of cells: hematoxylin-eosin staining and Nissl staining. All rats were anesthetized, perfusedand the complete brain wereremoved. Coronalsection of hippocampus and cortexwere collected and stained. The slides were captured under microscope to observe the pathological changes. The content of Nissl body was measured by Image J.3. We use the transmission electron microscope to observe the change of synaptic structure. All rats were anesthetized, perfused and the hippocampus CA1 were removed. Electron microscopic section were uranylacetate-plumbi nitras double stained and captured under transmission electron microscope. The changes of synaptic interface were measured by Image J.4. VGLUT1 m RNA level changes: real-time PCR. Total RNA in hippocampus and cortex were extracted by Trizol, and used β-actin as endogenous control to analyze the m RNA expression of VGLUT1.5. VGLUT1 protein level changes: western blottig. All protein in hippocampus and cortex were extracted, different kinds of protein were separated by SDS-PAGE. Then, the target protein were transferred to NC membrane,which will incubate with antibody later. Finally, we use an Chemiluminescent Method to detect the protein.6. We detect the Glu content in hippocampus by microdialysis combined with HPLC method to observe the effect of MA on Glu releasing. And then ininvestigate the effect of CSB6 B on Glu releasing induced by MA. Results:1. The effect of MA on learning and memery: Single and long-term(20d) intraperitoneal MA would cause avoidence memory and identification memory impairment in mice. At the early phase of long-term intraperitoneal MA could improve identification memory in rats, and had no effect at the later phase. MA could cause significant rewarding memory in CPP and behavioral sensitization in mice. These results indicated that influences of MA on learning and memory various from dose and time. Single injection can cause dose dependent impairment;long-term injection showed bidirectional effect. These results are similar to the excitement and cognitive dysfunction induced by MA.2. The result of hematoxylin-eosin staining showed that single and long-term intraperitoneal MA would lead to pathological changes in cells. The result of Nisslstaining showed that single and long-term intraperitoneal MA would cause Nissl body significantly loss in hippocampus and cortex. The result indicated that learning and memory impairment induced by MA is accompany with nerve injury.3. The result of transmission electron microscope showed that long-term intraperitoneal MA can decrease the number of synapse, broden width of synaptic cleft, lower the post-synaptic density, shorten the Length of synaptic active zone and decrease the interface curvature of synapse compare with the NS group. These changes of synaptic Structures indicated that long-term intraperitoneal MA can decrease synaptic transmission efficiency in hippocampus CA1 and provide material base of behavior changes.4. Both m RNA and protein expression of VGLUT1 were significantly decreased in hippocampus and cortex of mice compared with the NS group at the fifth day after single intraperitoneal MA, and the differences were disappeared at the fifteenth day. Both m RNA and protein expression of VGLUT1 were significantly increased in hippocampus and cortex of mice at the fifth day after the long-term intraperitoneal MA, and showed the same results at the fifteenth day. At the early phase of long-term intraperitoneal MA, m RNA expression of VGLUT1 were significantly decreased in hippocampus, and an opposite result in cortex compared with the NS group. The differences were disappeared at the later phase.5. MA can produce rewarding memory in rat. Our study showed that 1.0mg/kg and 2.0mg/kg MA could produce CPP in rat. And i.c.v. administering CSB6 B, a VGLUTs inhibitor, just prior to the administrationof MA was found to suppress theacquisitionof MA-induced CPP. Furthermore, i.c.v. administering CSB6 B would attenuates MA-induced behavioral sensitization induced in mice. These result indicated that regulate the concentration of Glu in synaptic cleft through inhibiting activity of VGLUT1 can block the rewarding memory of MA.6. Brain microdialysis and HPLC results showed that MA could increase extracellular Glu concentration. Compared with the basal level, extracellular Glu concentration increased 270% at 2.5h after MA injection. Pretreated with CSB6 B could decrease the extracellular Glu concentration in hippocampus, compared with MA group. These result indicated that inhibite the activity of VGLUT1 could decrease the Glu releasing induced by MA. Conclusion:MA exhibites different effects on learning and memory based on the treatment schedule and experimental design. MA-induced cognitive impairment is associated with dose and times of drug injection. Higher dose of MA produce more obvious learning and memory impairment; long-term treatment of MA showed bidirectional effect. MA cause nerve injury in hippocampus and cortex accompany with learning and memory impairment. VGLTU1 expression was changed along with the learning and memory impairment induced by MA. Inhibiting the activity of VGLUT1 blocked the development of MA-induced rewarding memory. These results indicated that the influences of MA on learning and memory was associated with expression and activity of VGLTU1.