The measurement of the extracellular L-Glu concentration in the m

The measurement of the extracellular L-Glu concentration in the medium was performed according to the methods previously

described (8). Real-Time Quantitative RT-PCR, Western Pictilisib chemical structure blotting, immunocytochemistry were also performed according to the methods previously described (8). The microglia culture was treated with LPS for 24 h in the presence or absence of antidepressants and the concentration of L-Glu in the medium was measured. All sets of the experiments were repeated in triplicate. All procedures described above were in accordance with institutional guidelines. In the previous report, we showed that the expression level of astrocytic L-Glu transporters was decreased Lumacaftor cell line in the astrocyte-microglia-neuron mixed culture in LPS (10 ng/ml, 72 h)-induced inflammation model without cell death (8). We first compared the effects of various groups of antidepressants, i.e., selective serotonin reuptake inhibitors (SSRIs) (paroxetine, fluvoxamine, and sertraline), serotonin–norepinephrine

reuptake inhibitor (SNRI) (milnacipran), and tricyclic antidepressant (TCA) (amitriptyline), on the decrease in the astrocytic L-Glu transporter function in this inflammation model. To quantify L-Glu transport activity, we measured the concentration of L-Glu remaining 30 min after changing the medium to the one containing 100 μM of L-Glu. In each set of experiment, LPS-induced decrease in the L-Glu transport activity was stably reproduced (Fig. 1A–E). Among antidepressants, only paroxetine prevented the LPS-induced decrease in L-Glu transport activity (Fig. 1A). The effect was concentration-dependent and reached significant at 1 μM. The other antidepressants had no effects (Fig. 1B–E). Typical image of the astrocyte-microglia-neuron mixed culture was shown in Fig. 1F. We have clarified that LPS-induced medroxyprogesterone decrease in L-Glu transport activity was caused by the decrease in the expression level of GLAST, a predominant L-Glu transporter in the mixed culture, in both of mRNA and protein levels (8). In this study, LPS-induced decreases in the

expression of GLAST, were reproduced at both of mRNA (28.8 ± 4.7% of the control) and protein (69.5 ± 4.7% of the control) levels (Fig. 1G, H). We then examined the effects of paroxetine on the LPS-induced decrease in the L-Glu transporter expression. Paroxetine significantly prevented the decreases at both of mRNA (28.8 ± 4.7 to 49.6 ± 3.3%; n = 10) and protein (from 69.5 ± 4.7% to 91.0 ± 5.1%; n = 5) levels ( Fig. 1G, H). As is shown in Fig. 1, fluvoxamine and sertraline, the other SSRIs in this study, did not affect the decrease in L-Glu transport activity, suggesting that paroxetine revealed the effects through the mechanisms independent of its inhibitory effect on serotonin selective transporter.

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