Figure 10.

Molecular mechanisms for the developmental regulation of Cx36. A, RT-qPCR analysis in rat hypothalamic cultures demonstrates that Cx36 mRNA expression increases during development and this increase is augmented by activation of group II mGluRs (LY379268) and inactivation of GABA_ARs (Bic/PiTX+A/C). However, it is not affected by inactivation of group II mGluRs (LY341495) and activation of GABA_ARs (muscimol). Relative Cx36 transcript levels are normalized to GAPDH, and normalized values are compared to DIV15 controls (set to 1.0). Statistical analysis was done using paired Student's t test relative to control (mean ± SEM). B, Dual-luciferase reporter assay. Cells were transfected on DIV3 with the luciferase (LUC) reporter plasmids driven by the rat Cx36 promoter (P_Cx36), incubated in the absence or in the presence of indicated agents and then assayed on DIV7. The NRSE deleted plasmid was produced using site-directed mutagenesis. The plasmid containing the Cx36 3′UTR was constructed by replacing the original SV40 poly A signal (SV40 pA) in the plasmid containing P_Cx36 with the full-length rat Cx36 3′UTR. Firefly luciferase values are normalized relative to Renilla luciferase values to control for transfection efficiency, and the results are presented as relative activity of the promoter constructs compared to the pGL3basic vector (set to 1.0). Statistical analysis was done using paired Student's t test relative to the corresponding nontreated control. Bic/PiTX+A/C, Bicuculline, picrotoxin, AP5, and CNQX.