Department of Biology Unit of Cell and Developmental Biology, University of Pisa, Pisa 56127, ItalyCenter for Neuroscience and Cognitive Systems, Istituto Italiano di Tecnologia, Rovereto, TN 38068, ItalyNeuroscience Institute, National Research Council (CNR), Pisa 56124, Italy
5-HT depletion in adult mice affects the density of serotonergic fibers that innervate the hippocampus and the thalamic PVN. A, Experimental timeline of TM treatment in Tph2GFP/fl°x::CMVCreERT mice. B, Representative images of raphe nuclei (top panel) and hippocampus (bottom panel) highlighting the depletion of serotonin within serotonergic cell bodies and fibers, respectively. C, Experimental timeline of serotonergic fiber density analysis. D–F, Representative coronal sections immunostained for GFP showing the distribution of serotonergic fibers in the dorsal hippocampus, ventral hippocampus, and in the thalamic PVN of vehicle- and TM-treated mice at D30. High-magnification images are shown on the right column of each panel. D’–F’, Quantification of serotonergic fiber density in the hippocampus and in the thalamic PVN of TM- and vehicle-treated mice. Quantitative analyses were performed by means of ROD measurements at D14 (n = 4 for each treatment), D30 (n = 7 for the hippocampus; n = 5 for the thalamic PVN), or D60 (n = 8 for the hippocampus; n = 6 for the thalamic PVN). Data are presented as percentage increase/decrease of ROD values of TM-treated mice as compared with vehicle-treated controls ± SEM. Wilcoxon singed rank test for paired data were used as test of significance. **p < 0.01; *p < 0.05; n.s., not significant, p > 0.05. Scale bar, 350 μm (B, top panels; D–F, left panels), 60 μm (B, bottom panels), 30 μm (D–F, right panels).
Alterations in the density of serotonergic fibers cannot be ascribed to changes in GFP expression in TM- versus vehicle-treated mice. A, Representative images showing the levels along the antero-posterior axis where GFP expression was analyzed (top row) and the 3D masks used to delimit the region in which the intensity of GFP immunosignal was measured (bottom row). B, Quantification of the intensity of GFP immunosignal in serotonergic neurons of TM- and vehicle-treated mice analyzed 30 days after the end of the treatment (n = 7 for each treatment). Analysis was performed using the Surface tool of the Imaris Bitplane software. C, Representative coronal sections immunostained for SERT showing the distribution of SERT-positive serotonergic fibers in the dorsal hippocampus, ventral hippocampus, and in the PVN of vehicle- and TM-treated mice analyzed 30 days after the end of the treatment. Data were analyzed using Wilcoxon signed rank for paired data. n.s., not significant, p > 0.05. Scale bar, 400 μm (A), 200 μm (C, dorsal hippocampus and ventral hippocampus), and 150 μm (C, PVN).
Serotonin depletion in TM-treated mice does not affect the hippocampal expression of BDNF and TrkB. A, Quantification of BDNF mRNA expression in the hippocampus of TM- and vehicle-treated mice 30 days after the end of the treatment. The quantification was performed by densitometric analysis of autoradiograms resulting from radioactive ISH experiments (n = 4 for each treatment). B, BDNF and TrkB protein expression level evaluated by Western blot analysis in the hippocampus of TM- and vehicle-treated mice (n = 3 TM-treated, n = 4 vehicle-treated mice) 30 days after the end of the treatment. GADPH and α-tubulin were used to normalize for variations on loading and transfer. Data are expressed as percentage of control (mean ± SEM). Student’s t test was used as test of significance. CA, Ammon’s horn; DG, dentate gyrus; n.s., not significant, p > 0.05.
5-HTP treatment restores serotonin immunoreactivity in the brain of Tph2GFP mutants. Representative images highlighting the reestablishment of 5-HT immunoreactivity within serotonergic neurons and fibers of Tph2GFP mutant mice following 24 h of 5-HTP treatment. Tph2GFP +/− animals treated with saline were used as controls. Scale bar, 350 μm (left panels) and 60 μm (right panels).
Reestablishing serotonin signaling in Tph2-deficient mice impacts on serotonergic fiber density and compensate alterations induced by 5-HT depletion. A, Experimental design of 5-HTP administration in TM-treated Tph2GFP/fl°x::CMVCreERT mice (left) and in Tph2GFP mutant mice (right). B, D, Representative confocal images illustrating the distribution and density of serotonergic fibers in the hippocampus and in the thalamic PVN. C, E, Quantification by ROD measurements of serotonergic fiber density in the hippocampus and the thalamic PVN. Data are presented as percentage increase/decrease of ROD values as compared with vehicle/saline-treated Tph2GFP/fl°x::CMVCreERT controls (n = 7 for treatment) or to saline-treated Tph2GFP +/− mice (n = 6 for treatment) ± SEM. Data were analyzed using the Friedman test followed by the Wilcoxon signed rank test and the Bonferroni’s correction was applied. ***p < 0.001; **p < 0.01; *p < 0.05; n.s., not significant, p > 0.05. Scale bar, 350 μm.
Computer-based mapping of serotonergic fibers and quantitative analysis of axon morphology. A, Representative high-magnification confocal acquisition in oblique view of GFP-immunoreactive serotonergic fibers (green) and computer-based 3D reconstructions (red). B, Graphs showing the quantification of the axon mean diameter and the ABP/AL ratio in Tph2GFP/fl°x::CMVCreERT (n = 5 for treatment) and Tph2GFP (n = 5 for treatment). Data were normalized to vehicle/saline-treated Tph2GFP/fl°x::CMVCreERT controls and saline-injected Tph2GFP +/− controls, respectively, and presented as mean percentage increase of controls ± SEM. C, Graphs showing the average tortuosity index (defined as the length of the axon divided by the Euclidean distance between the ends or branch points) of serotonergic fibers under the different experimental conditions. Data are presented as the mean ± SEM. Analysis of significance was performed using the Friedman test, followed by the Wilcoxon signed rank test. ***p < 0.001; **p < 0.01; *p < 0.05; n.s., not significant, p > 0.05. Scale bar, 4 μm.