Fig 2: General methodology. (A) The object location task (OLT) was used as a readout of hippocampal-dependent memory. Rats were submitted to restraint stress, followed by 3 min of training phase. After a delay of 5 min, the location of one of the objects (A’) was changed, and rats were tested for exploration time. (B) Dendritic spines were analyzed in CA1 secondary apical dendrites across the first 80 mm segments and total spines were classified as mature (mushroom-shaped) or immature (thin-spines, filopodia-like and stubby-shaped), as indicated in the left panel. Golgi stain was performed in the CA1 region of rat hippocampi (right panel), and white arrows indicate mature spines.(C) Morphological and biochemical characterization of the synaptoneurosome-enriched fraction. Electron micrograph showing a representative synaptoneurosome, characterized by the presence of an axon terminal identified by synaptic vesicles associated with a resealed postsynaptic compartment, and a postsynaptic density; evidencing intact synapses. Scale bar: 0.1 µm (left panel). Western blotting analysis of the different fractions obtained during the preparation of the synaptoneurosome-enriched fraction (H, homogenate; N, nucleus; S, synaptoneurosome). The presynaptic protein synapsin, the postsynaptic proteins PSD-95 and Homer-1, the a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA receptor subunits (GluA1 and GluA2) and the N-methyl-D-aspartate (NMDA) receptor subunits (NR1 and NR2B) were enriched in the synaptoneurosome fraction. The marker of glia component, GFAP, was distributed mainly in H and N.

Fig 3: Increased synaptic surface expression of AMPAR and basal excitatory transmission in hippocampal Fmr1R138Q neurons.a, b Super-resolution STED images of surface-expressed GluA1 and GluA2 (STED, green) in postsynaptic Homer1 sites (confocal, red) of TTX-treated WT and Fmr1R138Q hippocampal neurons. Scale bar, 500 nm. c, d Quantification of (a) and (b). Box plots indicate median (middle line), 25th, 75th percentile (box), and min to max values (whiskers) obtained for the mean surface GluA1 and GluA2 fluorescence intensity (c) and nanodomains (d) in WT and Fmr1R138Q neurons. N = 17–30 (WT) and 15–30 (Fmr1R138Q) neurons were analyzed from three biologically independent experiments with a total of dendritic spines analyzed ranging from 878 to 4246. Unpaired t test. ***p < 0.0001; **p = 0.0011. e–k mEPSCs recordings from acute hippocampal slices obtained from PND90 WT and Fmr1R138Q littermates. Quantification shows the mean values ± s.e.m. (e, g) and cumulative curves ± s.e.m. (f, h) of mEPSC amplitude and frequency. N = 10–11 neurons per genotype from three independent experiments. Unpaired t test. ns not significant. *p = 0.0111. i Example traces of WT and Fmr1R138Q single events. j Computed Tau rise and decay. k Representative traces of mEPSC recordings from WT and Fmr1R138Q hippocampal slices. Source data are provided as a Source Data file.

Fig 4: Effect of chronic stress on AMPA subunit levels in synaptosomal fraction of DH and VH. (A) Morphological characterization of synaptosomal fraction by electron microscopy. Photograph of the synaptosomal fraction; shows the presynaptic button and the presynaptic vesicles attached at the postsynaptic density. Picture was taken with 105,000× magnification. (B) Biochemical characterization of the different fractions obtained during synaptosome preparation. H represents the hippocampal homogenate, N represents nuclear fraction and Syn represents the synaptosomal fraction. Markers were evaluated by immunoblot. The nuclear marker (LAP-2a) was not detected in synaptosomal fraction. The PSD-95 (Post-synaptic marker), GluA1 and NR1 subunits of AMPA and NMDA receptor (NMDAR), respectively were enriched at the synaptosomal fraction. Levels of GluA1 and GluA2 subunit were evaluated in synaptosomal fraction obtained from dorsal (C) and ventral (D) hippocampus. Upper panel shows a representative western blot. Graphs represent mean ± SEM of each group. Number of animals is inside bars.

Fig 5: Increased surface expression of AMPAR in hippocampal neurons of Fmr1R138Q KI mice.a Immunoblots showing the levels of the indicated synaptic proteins in brain homogenates from PND21 WT and Fmr1R138Q littermate animals. GAPDH was used as a loading control. Quantification shows the mean ± s.e.m. of the total levels of the indicated proteins. N = 6 (GluA1, GluA2), N = 7 (PSD95), and N = 4 (GRIP1, PICK1, Stargazin) biologically independent experiments. Unpaired t test. ns not significant. **p = 0.0034. b Secondary dendrites from TTX-treated WT and Fmr1R138Q hippocampal neurons at 15 DIV stained for surface GluA1 (green) and MAP2 (red). Scale bar, 5 µm. Histograms show mean ± s.e.m. of both surface intensity and cluster density for GluA1 in WT and Fmr1R138Q neurons. Values were normalized to their respective basal conditions. N = 47 neurons per genotype from five biologically independent experiments. Two-tailed Mann–Whitney test. *p = 0.0147 (intensity) and *p = 0.0124 (density). c Immunoblots showing the surface expression of GluA1 and GluA2 in TTX-treated WT and Fmr1R138Q cultured hippocampal neurons at 15 DIV using biotinylation assays. Histograms show the mean ± s.e.m. of the normalized level of GluA1 and GluA2 subunits at the neuronal surface in WT and Fmr1R138Q neurons. N = 7 (GluA1) and 4 (GluA2) biologically independent experiments respectively. Two-sided ratio t test. **p = 0.0011 (GluA1); **p = 0.0074 (GluA2). d Immunoblots showing the basal surface expression of GluA1 and GluA2 in PND90 TTX-treated WT and Fmr1R138Q hippocampal slices using the BS3-crosslinking assay. Control tubulin immunoblot is included to confirm the absence of BS3 crosslinking intracellularly. The surface/intracellular ratio in the WT was set to 1 and Fmr1R138Q values were calculated respective to the WT. Error bars show the mean values ± s.e.m. N = 6 independent experiments. Two-tailed ratio t test. **p = 0.0041 (GluA1); **p = 0.0056 (GluA2). Source data are provided as a Source Data file.