The Role Of Glutamate Transporters In Methamphetamine-induced Nerve Injury

Methamphetamine (METH) is a widespread drug of abuse that cause dopamine (DA) neuronal apoptosis, severe ataxia, and learning and memory impairment, and make the ability of cognition and judge loss. METH-induced increases in extracellular concentrations of striatal DA and glutamate( Glu). Glu are known to contribute to the observed nerve terminal damage. METH increased striatal DA release via reverse transport, and increased striatal Glu concentration by the activation of the D1-associated striatonigral GABAergic pathway. Extracellular Glu levels are physiologically regulated by the activity of glutamate transporters and by the amount of Glu released from axon terminals.Since excitatory toxicity of Glu could be attenuated by regulating expression of glutamate transporters to decrease Glu concentration of synaptic cleft, could METH-induced nerve injury be diluted by the same way ? to solve this, the experiment proceed as follows:Objective:To investigate changes of expression of glutamate transporter 1(GLT1) and vesicular glutamate transporter 1 (VGLUT1) on METH-induced nerve injury in different brain regions; and to explore the possibility of relieving injury by regulating glutamate transporters; to study the correlation between glutamate transporters and DA,Glu,GABA transmitters release.Method:1. Establishment of acute, sub-acute animal model of nerve injury induced by METH. all rats were scored according to the stereotyped behaviors score standards. and the scores should greater than 2 points, which indicated that a successful model was founded.2. Nissl body were determined by Nissl staining method: after scoring stereotyped behavior, all rats were anesthetized and perfused, fixed dehydrated, and the brain were removed. according to the stereotaxic map, coronal section of cortex, hippocampus, striatum and nucleus accumbens were collected and patched on poly-lysine-coated slide separately. After staining, the slides were mounted by picric acid, dried overnight and observed under microscope, the nissl bodies would showed mottled blue (purple) staining;3. VGLUT1 and GLT1 mRNA level changes were detected by real-time PCR assay. Total RNA were Extracted by TRizol, and usedβ-actin as an endogenous control to analyze the mRNA expression of glutamate transporters;4. Changes of VGLUT1 and GLT1 expression were detected at the protein level: all protein were extracted, and the target protein were separated by SDS-PAGE, and the protein were transferred to NC membrane by electronic transfer of technology and incubation with antibody, then the target protein were detected by Chemiluminescent Method.5. To investigate the effect of METH on releasing neurotransmitters in different region. The concentration of Glu,Gly,and DA in striatum were detected by microdialysis combined with HPLC-FD method.Results:1. Exporsure to methamphetamine (5 mg·kg-1, 10 mg·kg-1, 20 mg·kg-1 and 40 mg·kg-1) could cause stereotypic behavior, such as repeated smelling and exploring, shaking and raising head in a dose-dependent manner. The scores of stereotypic behavior in rats showed that, compared to NS group, only 5 mg·kg-1 dose of stereotyped behaviors in rats less than 2 (1.4±0.24), and there was no significant difference; and the stereotyped behaviores scores of the other 3 groups ratswere (2.8±0.37), (3.2±0.2), (4.2±0.2) points respectively, and they had significant difference compared to NS group; In 40 mg·kg-1 group, stereotyped behaviores were most serious, and it had significant difference with 5,10,20mg·kg-1 group. So, in the following experiments, 40 mg·kg-1 was chosen as acute dose, and in sub-acute administration experiments METH dose was 10 mg·kg-1, because high dose of METH could leading death by successive administration. 2. Pretreated with ceftriaxone(200 mg·kg-1 ip) could significently inhibited stereotyped behavior induced by METH acute, sub-acute treatment, and the scores of METH groups rats were 3.7 times higher than that of NS group. Compared to METH group, pretreated with ceftriaxone could decrease the scores of stereotyped behavior by 67.5% significantly(P<0.05), which indicated that METH- induced stereotyped behavior could be extenuated by regulating expression of glutamate transporters.3. The result of Nissl staining showed that acute and subacute intraperitoneal METH would cause Nissl body significantly loss in the striatum. Pretreated with ceftriaxone could alleviate the nerve damage. The result proved that nerve injury induced by METH had close relationship with excitotoxicity, and reinforcing glutamate transporter function could improve nerve injury.4. VGLUT1 which located on the presynaptic vesicles membrane was responsible for the transport presynaptic Glu into the synaptic vesicles, and Glu was released into the synaptic cleft when some excitatory stimulus comes; GLT1 was located on the astrocytes cells and neurons membrane, its function was to uptake Glu in the synaptic cleft, and terminate its excitatory transmission. Western blot results showed that, after the acute administration of METH, compared with the saline control group, VGLUT1 protein expression was increased in striatum and nucleus accumbens by 66%, 102% respectively, and GLT1 protein expression was also significantly increased by 23%, 53.7% respectively. Pretreated with ceftriaxone (200mg·kg-1 ip) following by METH treatment could downregulate VGLUT1 protein expression in striatum and nucleus accumbens compared with METH group by 20%, 56% respectively; and GLT1 protein expression was upregulated by 15%, 36% respectively; The data was analysed by one-way ANOVA and SNK test, the result showed that each group had significant difference (P <0.05). The result showed that, ceftriaxone could attenuate toxicity of Glu by upregulating GLT1 protein expression and downregulaing VGLUT1 protein expression.5. Compared with the NS control group, sub-acute administration of METH significantly increased GLT1 protein expression in striatum, nucleus accumbens, prefrontal cortex and hippocampus by 60%, 40.9%, 28.3% and 68.6% respectively; and VGLUT1 protein expression in striatum and nucleus accumbens increased 66%, 52.9% respectively. VGLUT1 protein expression had no significent change in prefrontal cortex and hippocampus. Pretreated with ceftriaxone could reduced GLT1 protein expression at range 10% to 14%, and VGLUT1 protein expression in striatum was reduced by 20%.6. Compared with the NS group, sub-acute administration of METH significantly increased GLT1mRNA expression in striatum and hippocampus by 90%, 15% respectively, and in nucleus accumbens and prefrontal cortex, GLT1mRNA expression remained unchanged; and VGLUT1mRNA expression increased 160% in striatum, decreased 70% in nucleus accumbens, and it had no significent change in prefrontal cortex and hippocampus. Pretreated with ceftriaxone could down-regulate VGLUT1mRNA expression in striatum and hippocampus by 50%, 40% respectively, while incredsed VGLUT1mRNA expression in nucleus accumbens by 140%.7. Brain microdialysis and high performance liquid results showed: after acute administration of METH, extracellular Glu concentration wes increased continuously during the whole detection time(0-6 h), compared with the basal level, extracellular glutamate concentrations increased 450% at 5.5h after METH administration. And DA concentration reached peak level at 1h, compared with the basal level, extracellular DA concentrations increased 1248.6% at 1h. Compared to METH group, pretreated with ceftrixone significantly decreased the extracellular Glu concentration in striatum(P <0.05). The DA concentration at 1h was partly inhibited, but it still increased 838.4% compared with the basal level.Conclusion:METH acute and subacute administration caused nerve damage in different brain regions, and increased extracellular Glu concentrations, changed glutamate transporter GLT1 and VGLUT1 protein expression; Regulating glutamate transporters expression to decrease extracellular Glu concentrations could significantly dilute stereotyped behavior of rats and mitigate nerve injury. All above results proved that, neurotoxicity of METH was closely related to excessive release of excitatory amino acids, glutamate transporter may be a drug target for controlling METH-induced neurotoxicity.