Therefore, we analyzed the effect of MIP 2�� on GLT 1 and GLAST expression in astrocytes, as well as their redistribution into functional raft microdomains. We also measured changes in glutamate uptake in response to MIP 2�� overexpression and dissected the MIP 2�� sig naling pathways. Finally, we evaluated whether MIP 2�� overexpression enhanced neuronal sensitivity to glutam ate toxicity. Materials selleck kinase inhibitor and methods Expression vectors construction The mouse wild type MIP 2�� cDNA was subcloned into pAAV IRES hrGFP to create the MIP 2�� expression vector, pAAV MIP 2�� hrGFP. MIP 2�� cDNA sequences were confirmed by DNA sequencing. To construct an RNAi vector to si lence the MIP 2�� gene, we designed and synthesized three double stranded oligonucleotides targeting three different sites of the MIP 2�� cDNA that could generate hairpin small interfering RNAs.

Inhibitors,Modulators,Libraries The selection of the coding sequences for siRNA was analyzed by BLAST to ensure that they did not have significant sequence homology with other genes. Then, the oligonucleotides were inserted into pBS U6 according to the method of Sui et al. After confirmation by sequencing, Inhibitors,Modulators,Libraries RNAi vectors were transfected into astrocytes with Polyfect according to the manu facturers instructions. The negative control plasmid contains a scrambled sequence that does not show significant homology to mouse gene sequences. Cell culture and treatment Primary astrocytes were prepared from neonatal SJL J mouse brains using methods similar to those described previously.

Primary glial cell cultures were maintained in MEM supplemented with 10% FCS, 6 mg ml Inhibitors,Modulators,Libraries glu cose, and 5 ug ml bovine pancreas insulin, referred to as complete medium, in 10% CO2 at 37 C. After 11 days, the flasks were agitated on an orbital shaker for 14 hours at 250 rpm at 37 C, and the nonadherent oligodendrocyte and microglial cells were removed. Cortical astrocytes were purified from the primary mixed glial cell culture by three to four repetitions of trypsinization and replating. The purity of astrocytes was more than 95% when determined by indirect immunofluorescence using an anti glial fibrillary acid protein anti body. To increase surface expression of glutamate transporters, astrocyte cultures were treated with 250 uM dibutyryl cAMP for 7 days before experimentation. Neuron cultures were prepared from SJL J mice at em bryonic day 17.

In brief, the cortices were dissected and freed of meninges. Cortical fragments were incubated with 0. 25% trypsin and 20 ug ml DNase I in PBS at 37 C for 15 minutes. The cortical fragments were then disso ciated into single cells by pipetting, Inhibitors,Modulators,Libraries and the cells were suspended in Neurobasal A medium containing a B27 serum free supplement and plated Inhibitors,Modulators,Libraries onto poly D lysine coated plates. Twenty four hours later, not the cultures were treated with 5 uM cytosine arabinoside in vitro for 72 hours to prevent proliferation of other cell types.