Figure 1.
A: Euploid and Ts65Dn mice received a daily injection of either saline (Eu; Ts65Dn) or fluoxetine (Eu+Fluo; Ts65Dn+Fluo) from postnatal (P) day 3 to P15. Animals were killed on P45–P60 and used for evaluation of connectivity (P45) between the dentate gyrus and field CA3 and electrophysiological recordings from CA3 (P45–P60). B: Schematic drawing of a section across the hippocampal formation showing the major intrinsic connections. Patch clamp recording (rec) of miniature synaptic potentials were carried out from pyramidal neurons of field CA3. The area occupied by the mossy fiber terminals in the stratum lucidum of field CA3 is indicated in gray. C: Mossy fiber circuitry in CA3. Mossy fibers establish excitatory synapses (+) with pyramidal neurons and inhibitory interneurons in the stratum lucidum. Inhibitory interneurons establish inhibitory synapses (−) with the pyramidal neurons. Abbreviations: CA1-3, hippocampal fields; DG, dentate gyrus; IN, inhibitory interneuron; MF, mossy fivers; PN, pyramidal neuron; PP, perforant pathway; PYR, pyramidal layer; SCH, Shaffer collaterals; SL, stratum lucidum.
Figure 2.
Effect of fluoxetine on the thickness of the mossy fiber terminal field.
A–D: Examples of coronal sections, processed for PSA-NCAM immunohistochemistry, across the hippocampal formation of an animal from each of the following experimental groups: euploid (A), Ts65Dn (B), treated euploid (C), and treated Ts65Dn (D). Sections were taken at approximately the same distance from the rostral border of the hippocampal formation. The mossy fiber system (indicated by arrows) can be clearly recognized for its dark color and its abrupt termination at the border between CA3 and CA2. The scale bar = 300 µm applies to A–D. Abbreviations: DG, dentate gyrus; MF, mossy fibers. C: Mean thickness of the mossy fiber terminal field in CA3 in untreated euploid (n = 7), untreated Ts65Dn (n = 6), treated euploid (n = 6) and treated Ts65Dn (n = 4) mice. The mean thickness was obtained, from 3–4 sections per animal, by dividing the area occupied by the mossy fibers by the length of CA3. Values represent mean ± SD. * p<0.05; ** p<0.01 (Duncan's test after ANOVA).
Figure 3.
Effect of fluoxetine on overall innervation in stratum lucidum of field CA3.
A–B: Western blot analysis of synaptophysin levels in the hippocampal formation of untreated euploid (n = 5), untreated Ts65Dn (n = 5), treated euploid (n = 5) and treated Ts65Dn (n = 5) mice. Western immunoblots in (A) are examples from an animal of each experimental group. Histograms in (B) show synaptophysin levels normalized to GAPDH and expressed as fold difference in comparison with untreated euploid mice. C: Images of sections processed for synaptophysin immunofluorescence from field CA3 from an animal of each experimental group. Scale bar = 50 µm. D: Optical density of synaptophysin immunoreactivity in the stratum lucidum of untreated euploid (n = 7), untreated Ts65Dn (n = 6), treated euploid (n = 6) and treated Ts65Dn (n = 4) mice. Number of analyzed sections: 3–4 per animal. Six measurements were taken from each section in a box of 490 µm2, randomly placed in the stratum lucidum of CA3. Data are given as fold difference vs. the stratum lucidum of untreated euploid mice. E: Images, taken with the confocal microscope, of sections processed for synaptophysin immunofluorescence from the stratum lucidum of field CA3 from an animal of each experimental group. Scale bar = 5 µm. F: Number of puncta per µm2 exhibiting synaptophysin immunoreactivity in untreated euploid (n = 7), untreated Ts65Dn (n = 6), treated euploid (n = 6) and treated Ts65Dn (n = 4) mice. The density of individual puncta exhibiting synaptophysin immunoreactivity was evaluated in 3–4 sections per animal (image size; 512×512 pixels; three images per section). Values in B, D and F represent mean ± SD. ** p<0.01; *** p<0.001 (Duncan's test after ANOVA). Abbreviations: Eu, euploid; Fluo, fluoxetine; PYR, stratum pyramidale; RAD, stratum radiatum; SL, stratum lucidum.
Figure 4.
Effect of fluoxetine on the glutamatergic innervation in stratum lucidum of field CA3.
A–B: Western blot analysis of VGLUT1 levels in the hippocampal formation of untreated euploid (n = 5), untreated Ts65Dn (n = 5), treated euploid (n = 5) and treated Ts65Dn (n = 5) mice. Western immunoblots in (A) are examples from an animal of each experimental group. Histograms in (B) show VGLUT1 levels normalized to GAPDH and expressed as fold difference in comparison with untreated euploid mice. C: Images of sections processed for VGLUT1 immunofluorescence from field CA3 of an animal of each experimental group. Scale bar = 50 µm. D: Optical density of VGLUT1 immunoreactivity in the stratum lucidum of untreated euploid (n = 7), untreated Ts65Dn (n = 6), treated euploid (n = 6) and treated Ts65Dn (n = 4) mice. Number of analyzed sections: 3–4 per animal. Six measurements were taken from each section in a box of 490 µm2, randomly placed in the stratum lucidum of CA3. Data are given as fold difference vs. stratum lucidum of untreated euploid mice. E, F: Images, taken with the confocal microscope, of sections processed for double-labeling immunofluorescence with an anti-synaptophysin antibody (green) and an anti-VGLUT1 antibody (red) from the stratum lucidum of an animal from each experimental group. Images in (F) represent colocalization (white) between these two markers. Images correspond to the same images shown in Figure 3E. Scale bar = 5 µm. G: Coefficient of colocalization of synaptophysin (SYN) and VGLUT1 in stratum lucidum of untreated euploid (n = 7), untreated Ts65Dn (n = 6), treated euploid (n = 6) and treated Ts65Dn (n = 4) mice. The coefficient of colocalization was evaluated in 3–4 sections per animal (image size; 512×512 pixels; three images per section). Values in D and G represent mean ± SD. *** p<0.001 (Duncan's test after ANOVA). Abbreviations: Eu, euploid; Fluo, fluoxetine; PYR, stratum pyramidale; RAD, stratum radiatum; SYN, synaptophysin; SL, stratum lucidum.
Figure 5.
Effect of fluoxetine on CA3 pyramidal neuron spine density.
A: Photomicrograph of Golgi-stained field CA3 pyramidal cells showing the spinous excrescences on the proximal apical dendritic shaft in an animal from each experimental group. Scale bar = 10 µm. B: Density of spines on the proximal apical dendritic shaft in the stratum lucidum of field CA3 of untreated euploid (n = 9), untreated Ts65Dn (n = 5), treated euploid (n = 5) and treated Ts65Dn (n = 5) mice. Individual spines of the thorny excrescences were counted over the length of the dendritic shaft covered by spinous excrescences in 6–8 neurons per animal. Spine density is expressed as number of spines per 20 µm of dendrite. Values represent mean ± SD. * p<0.05; ** p<0.01; *** p<0.001 (Duncan's test after ANOVA).
Figure 6.
Effects of fluoxetine on mEPSC frequency in CA3 pyramidal neurons.
A: Exemplary current tracings recorded in the gap-free mode in four representative cells from untreated euploid and Ts65Dn mice and euploid and Ts65Dn mice treated with fluoxetine, showing mEPSC activity. Holding potential was −70 mV. Recordings were made in the presence of 1-µM TTx in the superfusing solution. B: Average frequency-distribution diagrams of mEPSC amplitude for untreated euploid and Ts65Dn mice (B1) and euploid and Ts65Dn mice treated with fluoxetine (B2). Numbers of observations are: 8 (euploid, untreated mice), 4 (Ts65Dn, untreated mice), 7 (euploid, fluoxetine-treated mice), and 5 (Ts65Dn, fluoxetine-treated mice) (these values indicate the number of animals of each group from which recordings were obtained; 2 to 4 cells were recorded from each animal). C: Average, overall mEPSC frequency in the four animal groups (n: as above). One-way ANOVA revealed the existence of a statistically significant difference among groups (p = 0.007). *, p<0.05 (Bonferroni post-test; the same test also revealed a significant difference, at the 0.01 level, between Ts65Dn, untreated mice and euploid, fluoxetine-treated mice).
Figure 7.
Effects of fluoxetine on mEPSCs due to mossy-fiber input to CA3 pyramidal neurons.
A–D: Average frequency-distribution diagrams of mEPSC amplitude for untreated euploid (A) and Ts65Dn (B) mice, and fluoxetine-treated euploid (C) and Ts65Dn (D) mice, before (open symbols) and during (filled symbols) application of the group-II metabotropic glutamate receptor (mGluR) agonist, DCG-IV (3 µM) plus 50-µM APV. The shadowed areas correspond to the frequency of the mEPSC activity removed by this drug treatment. Numbers of observations are: 7 (euploid, untreated mice), 4 (Ts65Dn, untreated mice), 6 (euploid, fluoxetine-treated mice), and 5 (Ts65Dn, fluoxetine-treated mice) (these values have the same meaning as explained in Fig. 8 legend). E: Average, overall frequency of the mEPSC activity removed by DCG-IV+APV treatment, obtained by subtraction, in the four animal groups (n: as above). One-way ANOVA revealed the existence of a statistically significant difference among groups (p<0.001). * and **, p<0.05 and 0.01, respectively (Bonferroni post-test; the same test also revealed a significant difference, at the 0.001 level, between Ts65Dn, untreated mice and euploid, fluoxetine-treated mice).
Figure 8.
Effects of fluoxetine on mIPSC frequency in CA3 pyramidal neurons.
Figure 9.
Effect of fluoxetine on hippocampal BDNF levels.
RNA expression levels of BDNF, quantified by RT-qPCR, in homogenates of the hippocampal formation from untreated euploid (n = 5), untreated Ts65Dn (n = 5), treated euploid (n = 5) and treated Ts65Dn (n = 4) mice. Data (mean ± SD) are given as fold difference in comparison with untreated euploid mice. p<0.05; *** p<0.001 (Duncan's test after ANOVA).
Figure 10.
Summary of the effect of fluoxetine on connectivity in the stratum lucidum.
A–D: Connectivity between the dentate gyrus (DG) and field CA3 in euploid mice (A), euploid mice treated with fluoxetine (B), Ts65Dn mice (C) and Ts65Dn mice treated with fluoxetine (D). Ts65Dn mice have a reduced number of granule cells (GC), mossy fibers (MF), glutamatergic terminals (GT) in the stratum lucidum and spines (SP) on the thorny excrescences (C). Treatment with fluoxetine rescues all these defects and restores the input from the DG to CA3 (D). In euploid mice treatment increases the glutamatergic terminals and spine density, with no effect on the number of granule cells and mossy fibers (B). The direction of the arrows indicates the direction of the defect and the effect of the therapy in comparison with untreated euploid mice. The double-headed arrows indicate no effect.