Figure 4.

Evaluation of survival and neurite outgrowth on hippocampal neuronal cells. (a) Cell viability was determined by the MTS assay (CellTiter 96A Aqueous One Solution Cell Proliferation Assay (Promega)) with hippocampal neurons after treatment with the galanin receptor 2 agonist (M1145, 100 nM) and Y1R receptor agonist (100 nM), either alone or in combination with or without the GAL 2 receptor antagonist (M871, 1 µM). Cells (20,000 per well) were seeded, and after seven days, the cells were incubated for 0, 8, 16, and 24 h in triplicates with the different groups. Blanks (medium only plus CellTiter 96A Aqueous One Solution Reagent) were subtracted from the value measured for each well incubated with the groups. M1145 and Y1R agonists’ incubation significantly increased neuronal survival in the dorsal DG. This effect was blocked by treatment with the GALR2 antagonist M871. * p < 0.05, 24 h after M1145 and Y1R agonist co-stimulation compared with every group according to Student’s unpaired t-test statistical analysis. Statistical values are presented in Supplementary Table S1. (b–d) GALR2 and Y1R agonists’ modulation of neurites’ outgrowth. Primary hippocampal neurons were treated for 24 h without (control) or with M1145 (100 nM) and/or Y1R agonists (100 nM) with or without the GAL 2 receptor antagonist (M871, 1 µM). The numbers of neurites per cell were determined after immunofluorescent labeling of neurons and neuronal nuclei (Pan Neuronal Marker (ABN2300)/neuronal nuclei (DAPI)). Quantification is shown in Figure 4b, where the data are presented as mean ± SEM. The combined group is significantly different from the rest of the groups (** p < 0.01 vs. the rest of the groups according to one-way ANOVA followed by Newman–Keuls post-hoc test). Statistical values are presented in Supplementary Table S1. Representative microphotographs of the significant increase in the number of neurites in the hippocampal cells after M1145 and Y1R agonist treatment (d) compared with the control group (c). The cells in green are hippocampal neuron-positive using confocal laser microscopy. White arrows point to neurite extensions. The nuclei are counterstained in blue by DAPI. Abbreviations: Control = Culture medium, M1145 = galanin 2 receptor agonist (100 nM), Y1R agonist = Y1R receptor agonist [Leu³¹-Pro³⁴]NPY (100 nM), M1145 + Y1R = co-administration of M1145 and Y1R, M1145 + Y1R + M871 = co-administration of M1145, Y1R, and GALR2 antagonist (1 µM).