Dopamine Receptor Has An Effect On The GABAa And NMDA Receptor In Salicylate-Induced Excitotoxicity Of Spiral Ganglion Neurons

Objective To investigate mRNA expression of dopamine receptor subtypes in the rat cochlear spiral ganglion neurons(SGN)following exposure to sodium salicylate.In addition,the effect of sodium salicylate on NR1 and GABRa2 subunit mRNA and surface/total protein expression under the circumstance of DR activation or blocking was analyzed.Methods Immunofluorescence techniques were applied to detect DR(DR1 and DR2)expression in cultured rat SGN.Moreover,RT-PCR and Western-blot were performed to assess NR1 and GABRa2 subunit mRNA and surface/total protein expression of primary cultured rat SGN after treatment of sodium salicylate as well as DR agonists or antagonists.Results(1)Immunofluorescence images showed co-localization of DR1/DR2 and βⅢ-tubulin protein in SGN bodies and axons.(2)RT-PCR results illustrated that ① DR subtypes of D1-5 were detected in the SGN.② The mRNA expression of all subtypes of DR and GABRa2,NR1 subunits were obviously upregulated except subtype D3 after treatment with sodium salicylate.Among them,D1 expression was increased 34.64%(t =-5.123,p =0.007<0.05),D2 expression was increased 34.60%(t =-5.206,p =0.006<0.05),D4 expression was increased 20.87%(t =-3.337,p = 0.029<0.05),D5 expression was increased 26.42%(t =-6.054,p = 0.004<0.005),GABRa2 expression was increased 30.41%(t =-2.839,p = 0.047<0.05),NR1 expression was increased 39.22%(t =-6.243,p= 0.003<0.005).③After exposure to sodium salicylate(5 mM),dopamine(100 μM),DR1 agonist(SKF38393,20 μM),DR2 agonist(quinpirole,20 μM),GABRa2 expression were increased 21.78%,27.45%,33.02%,33.42%respectively,and NR1 expression were.increased 28.70%,26.82%,29.03%,35.05%.④ Compared with the group of sodium salicylate treatment alone,both sodium salicylate + DR1 antagonist(SCH23390,20 μM)group and sodium salicylate + DR2 antagonists(Eticlopride,20 μM)group had a suppression on GABRα2 and MR1 mRNA expression.GABRα2 mRNA was reduced 29.56%,37.10%,and NR1 mRNA expression was decreased 37.62%,32.83%respectively.(3)Western-blot results demonstrated "that ①After treatment with sodium salicylate,surface protein expression of GABRa2 and NR1 were significantly decreased,GABRa2 fell 44.69%,NR1 was reduced by 21.57%,while the total protein of GABRa2 and NR1 had no significant change.②GABRa2 and NR1 surface protein expression of both the sodium salicylate +DR1 antagonist group and sodium salicylate + DR2 antagonist group had no significant difference compared with the control group.Conclusion(1)Most of the DR subtypes mRNA expression in SGN were increased following exposure to sodium salicylate.(2)Sodium salicylate significantly regulated the surface GABAaR and NMDAR trafficking on SGN,and the influence on GABAaR was more obvious.(3)DR may be involved in the effect of sodium salicylate on GABAaR and NMDAR through mediating mRNA expression as well as surface receptors trafficking.