Fig 2: Nuclear and post-synaptic levels of the m6A modifying proteins do not change upon training in symptomatic Hdh+/Q111 (KI) mice. Western Blots for METTL14 and FTO in A–C nuclear enriched fractions, D, E post-synaptic (PSD) and F, G non-PSD fractions from hippocampus of 8-month-old mice subjected to the training in the OLT. A–C Nuclear fraction was normalized to Histone H3. Two-way ANOVA reported a significant training effect in FTO levels (F(1,25) = 10.96, p = 0.0028) and in METTL14 levels (F(1,26) = 7.101, p = 0.0131) (n = 6–10 animals/condition). Right: Representative immunoblots D, E PSD fraction was normalized to PSD95. Two-way ANOVA reported a significant training effect in FTO levels (F(1,15) = 11.21, p = 0.0044) (n = 4–6 animals/condition). Below the graphs: Representative immunoblots (F, G). Non-PSD fraction was normalized to synaptophysin. (n = 5–7 animals/condition). Below the graphs: Representative immunoblots. Data were analyzed using Tukey as a post hoc test. *p < 0.05 and **p < 0.01 compared with WT naive mice. $p < 0.05 compared with WT-trained mice. Data are presented as mean ± SEM. N naïve, T trained, Syn synaptophysin

Fig 3: cAT data.Three 1.277 μ m ×1.186 μ m images showing conjugate EM and IF data. The left panel has synapsin (green) and PSD-95 (red) data overlaid, marked by the colored boundary lines. The scale bar represents 500 nm. The presence of both presynaptic and postsynaptic channels indicates the presence of a synapse with high probability. The center panel shows the PSD-95 IF image and the right panel shows the synapsin IF image. On both images, the EM-identified synaptic cleft is marked by a blue box. While the proposed synapse detection method uses multiple synaptic markers, only two are shown here for visualization purposes.

Fig 4: Synapse detection results.Top Left: Histogram of a slice of the probability map. Top Middle: Thresholded probability map, see text for details on the threshold selection. Top right: Probability map of one slice of a 13 μ m ×17 μ m region of the AT data on [19]. Bottom Row:. A series of pseudo-color images indicating the presence of the receptors PSD-95 (red), VGluT1 (blue), and synapsin (green) across three consecutive slices. Centroids of detected synapses containing all three receptors (query) are circled in black.

Fig 5: Synaptic density in microglial SIRPa deficient mice is lower on account of increased microglial engulfment of synaptic structures.a Schematics of the experimental procedures. IF immunofluorescence, mEPSC miniature excitatory postsynaptic current, TAM tamoxifen, P postnatal. Neonatal mice were injected 50 µg tamoxifen at P1, P2, and P3. b, c Representative confocal images depict synaptic staining for presynaptic marker Vglut1 (green) and postsynaptic marker Homer1 (red) in primary visual cortex (V1) (b) or hippocampal CA1 stratum radiatum (c) in SIRPafl/fl (+TAM), Cx3cr1CreERT2:SIRPafl/fl (-TAM), and Cx3cr1CreERT2:SIRPafl/fl (+TAM) mice at different time points. Synaptic number was quantified as colocalized pre- and postsynaptic puncta. Scale bar, 10 µm. d–i Histograms depict synaptic density in V1 (d–f) or hippocampal CA1 (g–i) in those mice. n = 5 mice/group; average of 10–12 fields from each mouse. One-way ANOVA analysis with Dunnett’s multiple comparisons test. j Example traces of mEPSCs (miniature excitatory postsynaptic currents) from SIRPafl/fl (+TAM), Cx3cr1CreERT2:SIRPafl/fl (-TAM), and Cx3cr1CreERT2:SIRPafl/fl (+TAM) mice. k, l Histogram shows average mEPSC frequency (k) and amplitude (l) of SIRPafl/fl (+TAM), Cx3cr1CreERT2:SIRPafl/fl (-TAM), and Cx3cr1CreERT2:SIRPafl/fl (+TAM) mice. n = 14, 12, 13 cells/group from 4 to 5 mice. One-way ANOVA analysis with Dunnett’s multiple comparisons test. m, n Cumulative probability distributions of mEPSC inter-event intervals (m) and amplitude (n) of SIRPafl/fl (+TAM), Cx3cr1CreERT2:SIRPafl/fl (-TAM) and Cx3cr1CreERT2:SIRPafl/fl (+TAM) mice. o, p Three-dimensional reconstruction and surface rendering demonstrate larger volumes of PSD95 puncta inside Iba-1 positive microglia in V1 from P28 Cx3cr1CreERT2:SIRPafl/fl (+TAM) mice. n = 7 mice/group, average of 8–9 microglia from each mouse, one-way ANOVA analysis with Dunnett’s multiple comparisons test. Grid line increments = 5 µm. Data are mean ± s.e.m. **P < 0.01, ***P < 0.001, NS not significant. Detailed statistical information was listed in Supplementary Statistical Data. Source data are provided as a Source Data file.