FIGURE 3.

GALR2/Y1R heteroreceptor complexes are detected by in situ PLA in the dorsal dentate gyrus (DG). (A) The diagram shows the presence of positive red PLA signals (red circles) mainly in the subgranular zone (Sgz) of the dentate gyrus at the border between the granular cell layer (Gcl) and polymorphic layer (P) of the dentate gyrus in the dorsal hippocampus. PLA positive red signals were also observed in the polymorphic layer. Blue-filled circles indicate a negative PLA signal in the molecular layer (M). [Bregma: –3.6 mm; according to the Paxinos and Watson (1998) stereotaxic atlas]. (B) Quantification of PLA signals in Sgz was performed by measuring red PLA positive blobs per nucleus per sampled field by an experimenter blind to treatment conditions. Sprague Dawley rats significantly increased GALR2-Y1R heterocomplexes (PLA blobs) in the Sgz after M1145 and Y1R agonist intranasal administration. This effect was blocked by treatment with the GALR2 antagonist M871. ***p < 0.001 vs. control and M1145; ★★ p < 0.01 vs. Y1R and M1145 + Y1R + M871 according to one-way ANOVA (F4, 15 = 10.21, p < 0.001) followed by Newman-Keuls post-hoc test. Inter-group comparisons are indicated by the vertical lines from the horizontal line above bars. Data are expressed as mean ± SEM, four rats per group, duplicates). (C–F) Representative microphotographs of the significant increase in the density of GALR2-Y1R positive red PLA blobs in the Sgz sub-region after GAL and Y1R agonist coinjection (E) compared with the control group (C). Magnified views from dashed boxes in (C,E) are shown in (D,F), respectively. GALR2-Y1R heteroreceptor complexes are shown as red PLA blobs (clusters) found in high densities per cell in many nerve cells using confocal laser microscopy. White arrows point to PLA positive clusters. Dashed lines outline the Gcl of the dentate gyrus. The nuclei are shown in blue by DAPI. Abbreviations: Control = Distilled water; M1145 = Galanin 2 receptor agonist 132 µg; Y1R = Y1R receptor agonist [Leu³¹-Pro³⁴]NPY 132 µg; M1145 + Y1R = Coadministration of M1145 and Y1R; M1145 + Y1R + M871 = Coadministration of M1145, Y1R and GALR2 antagonist M871 132 µg. Treatments were performed 24 h before brain processing; see material and methods for further details.