In situ proximity ligation assay and analysis of neurite length
To study the GALR2-Y1R heteroreceptor complexes, the in situproximity ligation assay (in situ PLA) was performed as described previously (Borroto-Escuela et al., 2021; Narváez et al., 2021). After permeabilization with PBS containing 0.2% Triton X-100 for 5 min, cells were treated with PBS containing 1% bovine serum albumin. The hippocampal cells were then incubated with the primary antibodies diluted in a suitable concentration in the blocking solution at 4 °C overnight. Then, cells were washed twice, and the proximity probe mixture (Duolink PLA probe anti-goat MINUS and Duolink PLA probe anti-rabbit PLUS, Sigma-Aldrich, Stockholm, Sweden) was applied to the samples and incubated for 1 h at 37 °C in a humidity chamber. The unbound proximity probes were removed by washing the slides twice, 5 min each time, with blocking solution at room temperature under gentle agitation and the sections were incubated with the hybridization-ligation solution (BSA, 250 g/ml), T4 DNA ligase (final concentration of 0.05 U/μl), 0.05% Tween-20, 250 mM NaCl, 1 mM ATP and the circularization or connector oligonucleotides (125–250 nM)) and incubated in a humidity chamber at 37 °C for 30 min. The excess of connector oligonucleotides was removed by washing twice, for 5 min each, with the washing buffer A (Sigma-Aldrich, Duolink Buffer A (8.8 g NaCl, 1.2 g Tris Base, 0.5 ml Tween 20 dissolved in 800 ml high purity water, pH to 7.4) at room temperature under gentle agitation and the rolling circle amplification mixture (Duolink amplification red, DUO82011, Sigma-Aldrich, Stockholm, Sweden) was added to the cells and incubated in a humidity chamber at 37 °C for 100 min. Then, the cells were incubated with the detection solution in a humidity chamber at 37 °C for 30 min. In a last step, the cells were washed twice in the dark, for 10 min each, with the washing buffer B (Sigma-Aldrich, Duolink Buffer B (5.84 g NaCl, 4.24 g Tris Base, 26.0 g Tris-HCl. Dissolved in 500 ml high purity water, pH 7.5) at room temperature under gentle agitation. The coverslips were put on a microscope slide and a drop of appropriate mounting medium (e.g., Duolink Mounting Medium, Sigma-Aldrich) was applied and sealed with nail polish. The slides were protected against light and stored for several days at −20 °C before confocal microscope analysis. The in situ PLA experiments were performed using the following primary antibodies: rabbit anti GALR2 (Alomone Lab, 1:100) and goat anti NPYY1R (sc-21992 Santa Cruz Biotechnology INC, CA, 1:200). Furthermore, cells were labelled with Neuro-Chrom Pan Neuronal Marker primary antibody (ABN2300, 1:100, Sigma-Aldrich; Merck Life Science S.L.U.) for 1 h, extensively washed, and stained with the green fluorescence secondary antibody goat anti-rabbit DyLight 488 (Jackson Laboratories InmunoResearch, 1:100). Cell nuclei were counterstained with DAPI (blue) contained in the mounting medium. The negative control consists in the omission of the species-specific primary antibody corresponding to the GALR2 in the presence of the two PLA probes. As a positive control of the PLA assay, a parallel analysis of the 5-HTR1A-5HTR2A isoreceptor complexes has been performed as previously documented (Borroto-Escuela et al., 2017). Acquisition of microscopy images, In situ PLA data analysis and morphometric quantifications were performed as previously described (Narvaez et al., 2020).