Studies On The Expression Of Amino Acid Transporters In The Brain Of Pentylenetetrazole Kindled Epileptic Rats And Its Correlation With BDNF

Objectives1.To study the expression changes of gamma-aminobutyric acid transporter-1 (GAT-1),glutamate transporter-1(GLT-1) and phosphorylated -tyrosine kinase receptor (p-Trk) in the brain of pentylenetetrazole(PTZ)-kindled chronic epileptic rats.2.To investigate the effect of brain-derived neurotrophic factor(BDNF) on the GLT-1 expression and its function in cultured astroglial cells,as well as the expression of p-Trk. So as to address the relationship between BDNF and amino acid transporters and its relevant functional significance in the chronic epilepsy's process.Methods and Results1.A rat kindling model of epilepsy was established by PTZ-injection.Then the occurrence and seizure level was evaluated by using Racine's method.The rats withâ…¢toâ…¤seizure level were chosen for the following studies.The success and survival rate is above 90%.2.The GAT- 1 and GFAP expression in the brain of PTZ-kindled rats was observed by immunohistochemistry at different phases(3,7,14d).The image was analyzed by Image-Pro software and the difference between control and kindled groups was determined by the ratio of positive-area.The results suggested that the expression of GAT-1 and GFAP increased at different level in the kindled rats,and the GAT-1's ratio between kindled and control was obviously higher than that of GFAP.3.The expression of GAT-1,GLT-1,GFAP and p-Trk in the hippocampus and cortex of the kindling rats(7d) was determined by Western blot.The results showed that all the expression of GAT-1,GLT-1,GFAP and p-Trk increased markedly comparing with that of the control,and the GAT-1's ratio between kindled and control was obviously higher than the other molecules.It implies there are significant changes of amino acid transporters after the occurrence of epilepsy,and there's other source for GAT-1 up-regulation except astrocyte.4.The co-localization of the p-Trk and GFAP in cortex and hippocampus of kindled rats was observed using double labelling immunofluorescence,so as to investigate the relation between p-Trk and astrocyte.The result showed that the double-positive(GFAP and p-Trk) cells were with astroglial shape and increased apparently in the kindling-group(7d).5.Astroglial cells from rat cerebral cortex were cultured and purified in vitro.The astrocyte was identified by GFAP indirect immunofluoresence.The rate of GFAP-positive cells in the cultured system was above 95%.6.The effect of different concentration of BDNF(50,100,200ng/l) on the expression of GLT-1 and p-Trk in cultured astrocytes was investigated by using Western blot method at different phases(1,3,7,14d).The BDNF treated cells showed an increase of GLT-1 and p-Trk expression when comparing with the control.The most effective concentration of BDNF was 100ng/l.The most significant expression increase in GLT-1 was at the 3d while p-TrkB at the 7d.It suggested that BDNF can enhance the expression of GLT-1 in astrocyte, and it is possibly related with the BDNF/TrkB pathway.7.By adding DL-[2,3-³H]-glutamate into the culture system of astrocyte treated with BDNF,the content of DL-[2,3-³H]-glutamate in the astroglial cells was measured by liquid scintillation counter.The result showed that the content of DL-[2,3-³H]-glutamate in BDNF-treated group was much higher than that of the control.The most effective concentration of BDNF is 100ng/l.It demonstrated that BDNF can enhance the glutamate-uptaking ability of astrocyte.8.The effect of BDNF on the[Ca²⁺]i activity in cultured astrocytes was examined by calcium imaging under the confocal laser microscope.It was found that BDNF could cause a gradual rise in the cytoplasmic[Ca²⁺]i concentration of astrocyte,suggesting BDNF executes through[Ca²⁺]I related signal pathways.ConclusionIn summary,the present study indicated that the expression of GAT-1,GLT-1 and p-Trk was up-regulated after epileptic seizure.BDNF can enhance the expression as well as its uptake function of GLT-1 in cultured astroglial system through the BDNF/TrkB pathway and is possibly related with Ca²⁺ mobilization.So BDNF may participate the neural plasticity by modulating glial amino acids transporters.Its up-regulation in epilepsy may have the effect antagonizing and recovering the excitable change caused by neurotransmitter imbalance.