Figure 5
GPR37 deletion bolsters striatal A2AR cell surface expression and function. (A) Coronal brain slices (300 µm) from GPR37+/+ and GPR37−/− mice were prepared and biotinylated as described in Material and Methods section. Total and cell surface extracts were analyzed by SDS-PAGE and immunoblotting as described in Fig. 2. (B) Quantification of A2AR cell surface density in GPR37+/+ and GPR37−/− striatum. Cell surface density was normalized using the total density of A2AR and expressed as mean ± SEM from six independent experiments. The asterisk indicates statistically significant difference from the control condition (P < 0.05; paired Student’s t test). (C) Co-distribution of GPR37 and A2AR in striatal synaptosomes. Immunofluorescence detection of GPR37 (red) and A2AR (green) in striatal total synaptosomes was performed as described in Materials and Methods. Superimposition of images (merge) reveals co-localization in yellow (arrows). (D) Quantification of synaptosomes expressing GPR37 and/or A2AR. The data are expressed as the percentage (mean ± SEM) of total number of synaptosomes that are endowed with GPR37 and/or A2AR, quantified in 3–4 different synaptosomal preparations from different mice, in which four different fields acquired from two different coverslips were analysed in each preparation. (E) A2AR-mediated cAMP accumulation in synaptosomes. Total striatal synaptosomes from GPR37+/+ and GPR37−/− mice were stimulated with 500 nM CGS21680 for 30 min at 37 °C and the cAMP accumulation was measured as described in Materials and Methods. The data are expressed as percentage (mean ± SEM) of forskolin-induced cAMP accumulation from eight independent experiments, which was similar (P > 0.05) in both genotypes. The asterisks indicate statistically significant difference from the control condition (P < 0.01; Student’s t test). (F) Co-distribution of GPR37 and A2AR in striatal neurons. Immunofluorescence detection of GPR37 (red) and A2AR (green) in primary cultured striatal neurons (DIV21) was performed as described in Materials and Methods. Superimposition of images (merge) reveals co-localization in yellow. Scale bar: 100 μm. (G) A2AR-mediated cAMP accumulation in striatal neurons. Striatal primary neurons from GPR37+/+ and GPR37−/− were stimulated with 500 nM CGS21680 for 30 min at 37 °C and the cAMP accumulation was measured as described in Materials and Methods. The data are expressed as percentage (mean ± SEM) of forskolin-induced cAMP accumulation from five independent experiments, which was similar (P > 0.05) in both genotypes. The asterisks indicate statistically significant difference from the control condition (P < 0.01; Student’s t test).
