Rat Slc1a3 (Excitatory amino acid transporter 1) ELISA Kit

Objective Abnormalities in neurotransmission within the cortico-striatal-thalamo-cortical circuitry are implicated in the pathogenesis of Tourette syndrome. Glutamate is a major excitatory neurotransmitter and an important member in the cortico-striatal-thalamo-cortical circuitry. To explore the role of glutamatergic neurotransmission in genetic susceptibility of Tourette syndrome, we carried out the genetic and functional characterization of sequence variants in SLC1A3 gene, which encodes the main glutamate transporter in astrocytes in individuals with well-characterized Tourette syndrome (n = 256) and normal controls (n = 224). Methods Exon-containing regions of SLC1A3 gene were screened using capillary electrophoresis-single strand conformation polymorphism followed by direct sequencing. Sequence variants were genotyped by restriction enzyme digestion and studied using glutamate uptake assay and membrane protein pull-down for transporter function. Results A missense variant involving a highly conserved residue, E219D, was identified in 11 heterozygous individuals with Tourette syndrome and four in the controls. The allele frequency for E219D was 2.4 folds higher in the Tourette syndrome (0.022) compared with the control cohort (0.009) although the difference did not reach statistical significance in the current cohorts (P = 0.09). A H-3-glutamate-uptake assay showed that E219D conveys a significant increase (1.66 fold) in the SLC1A3-mediated glutamate uptake in HEK293 cells. A biotin-mediated membrane pull-down analysis showed a similar increase (1.5 fold) of mutant SLC1A3 protein in the membrane fraction of transfected HEK293 cells compared with that in the wild type controls. Conclusion These results indicate that E219D is a functional SLC1A3 variant that is presented in a small number of individuals with Tourette syndrome. Further studies on possible changes in glutamate transport in the pathogenesis of Tourette syndrome are warranted. Psychiatr Genet 21:90-97 (C) 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins.

Purpose: This study investigated changes in the transcript levels of genes related to glutamate neurotransmission and transport as diabetes progresses in the Long-Evans rat retina. Transcript levels of vascular endothelial growth factor (VEGF), erythropoietin, and insulin-like growth factor binding protein 3 (IGFBP3) were also measured due to their protective effects on the retinal vasculature and neurons. Methods: Diabetes was induced in Long-Evans rats with a single intraperitoneal (IP) injection of streptozotocin (STZ; 65 mg/kg) in sodium citrate buffer. Rats with blood glucose >300 mg/dl were deemed diabetic. Age-matched controls received a single IP injection of sodium citrate buffer only. The retinas were dissected at 4 and 12 weeks after induction of diabetes, and mRNA and protein were extracted from the left and right retinas of each rat, respectively. Gene expression was analyzed using quantitative real-time reverse-transcription PCR. Enzyme-linked immunosorbent assay was used to quantify the concentration of VEGF protein in each retina. Statistical significance was determined using 2x2 analysis of variance followed by post-hoc analysis using Fisher's protected least squares difference. Results: Transcript levels of two ionotropic glutamate receptor subunits and one glutamate transporter increased after 4 weeks of diabetes. In contrast, 12 weeks of diabetes decreased the transcript levels of several genes, including two glutamate transporters, four out of five N-methyl-D-aspartate (NMDA) receptor subunits, and all five kainate receptor subunits. Diabetes had a greater effect on gene expression of NMDA and kainate receptor subunits than on the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor subunits, for which only GRIA4 significantly decreased after 12 weeks. VEGF protein levels were significantly increased in 4-week diabetic rats compared to age-matched control rats whereas the increase was not significant after 12 weeks. Transcript levels of VEGF and VEGF receptors were unchanged with diabetes. Erythropoietin and IGFBP3 mRNA levels significantly increased at both time points, and IGFBP2 mRNA levels increased after 12 weeks. Conclusions: Diabetes caused significant changes in the transcriptional expression of genes related to ionotropic glutamate neurotransmission, especially after 12 weeks. Most genes with decreased transcript levels after 12 weeks were expressed by retinal ganglion cells, which include glutamate transporters and ionotropic glutamate receptors. Two genes expressed by retinal ganglion cells but unrelated to glutamate neurotransmission, gamma-synuclein (SNCG) and adenosine A1 receptor (ADORA1), also had decreased mRNA expression after 12 weeks. These findings may indicate ganglion cells were lost as diabetes progressed in the retina. Decreased expression of the glutamate transporter SLC1A3 would lead to decreased removal of glutamate from the extracellular space, suggesting that diabetes impairs this function of Muller cells. These findings suggest that ganglion cells were lost due to glutamate excitotoxicity. The changes at 12 weeks occurred without significant changes in retinal VEGF protein or mRNA, although higher VEGF protein levels at 4 weeks may be an early protective response. Increased transcript levels of erythropoietin and IGFBP3 may also be a protective response.