GALR2 agonist and Y1R agonist interaction enhanced GALR2/Y1R heteroreceptor complexes and neurite length on hippocampal neuronal cells
To study at receptor level the cellular mechanisms related to the observed in vivo effects we performed in situ proximity ligation assay (PLA) on hippocampal neuronal cells. This procedure allowed to analyze the GALR2/Y1R heteroreceptor complexes formation after M1145 and/or Y1R agonist incubation.
PLA-positive red clusters were found specifically in the membrane and cytoplasmatic region of hippocampal neuronal cells (Figure 4a-d). Quantification of PLA demonstrated an increase in the density of the PLA-positive red clusters after Y1R agonist incubation compared to control (one-way ANOVA, F4,20 =16.25 , p < 0.001, Newman-Keuls post-hoc test: p<0.05) or M1145 incubation (Newman-Keuls post-hoc test: p<0.05) (Figure 4 a,c-d). Moreover, upon incubation with M1145 and the Y1R agonist significantly increased the number of PLA-positive red clusters in the hippocampal neuronal cells (Figure 4a, d) compared to control (Newman-Keuls post-hoc test: p<0.001), M1145 alone (Newman-Keuls post-hoc test: p<0.001) and Y1R agonist alone (Newman-Keuls post-hoc test: p<0.01). Moreover, the specific GALR2 antagonist M871 counteracted this synergic effect (Newman-Keuls post-hoc test: p<0.01) (Figure 4a), demonstrating that this interaction was mediated through the coactivation of GALR2 and Y1R.
Due to the changes observed in the density of these complexes upon treatments, we decided to study the morphology and structural plasticity changes of hippocampal cultures upon these pharmacological treatments. The data show a significant synergistic increase of mean neurite length upon coactivation of M145 and the Y1R agonist for 24 hours compared to the YR1 agonist group alone (one-way ANOVA, F4, 20 = 16.91, p < 0.001, Newman-Keuls post-hoc test: p<0.05), control (Newman-Keuls post-hoc test: p<0.001) and M1145 alone (Newman-Keuls post-hoc test: p<0.001) (Figure 4b,c,d). The Y1R agonist incubated alone increased the neurite length compared to control and M1145 alone (Newman-Keuls post-hoc test: p<0.01) (Figure 4b). Furthermore, the presence of GALR2 antagonist M871 entirely blocks the synergic effects (Newman-Keuls post-hoc test: p<0.05) (Figure 4b).