Fig 2: IL-4Ra pathway in the presynapse. (A–D) Exemplary images of STED microscopy for IL-4Ra and synaptic markers in hippocampal stratum radiatum of adult mice. Representative images of two independent experiments. Boxed areas are enlarged for improved visualization. (A) IL-4Ra (green) colocalizes (arrows) with presynaptic Syn1 (red). (B) IL-4Ra is in close proximity (arrow) to the active zone protein Bsn (red), but not to Homer-1 (magenta, arrowhead). (C) Inhibitory synapses marked by VGAT (red) also express IL-4Ra (arrows). (D) IL-4Ra expression in excitatory synapses marked by VGLUT1 (red, arrows). (E–G) STED microscopy in human postmortem brain tissue shows overlap (arrows) of IL-4Ra (green) with (E) Syn1 (red), (F) VGLUT1 (red), and (G) VGAT (red). (H) Schematic summary of presynaptic IL-4Ra localization. (I) Quantification of colocalization of IL-4Ra with VGLUT and VGAT in murine stratum radiatum. (J) ImmunoEM of synapse in stratum radiatum of Balb/C WT (The Jackson Laboratory) showing expression on the membrane (arrows) and vesicles (arrowhead). (K) Expression is abolished in Balb/C.il4ra-/- synapse. Boxed areas (1–4) are enlarged for improved visualization. Representative images of three independent experiments. (L) Co-IP with anti–IL-4Ra or isotype IgG control on synaptosomes shows presence of GAP-43 (~50 kD) in the presynaptic signaling pathway. Input represents total synaptosome lysate; output corresponds to the co-IP product; GAP-43 was absent in isotype control. Based on three independent experiments. (M) Representative Western blot (three independent experiments) of pPKC? (~90 kD) and ß-actin (~40 kD) for PBS and IL-4–treated ultrapure synaptosomes. (N) Quantification shows induction of PKC? phosphorylation by IL-4 (n = 6) compared to PBS (n = 5). (O) Schematic representation of IL-4R signaling pathway in presynapse. (P) Quantification of IL-4 expression relative to RPS29, normalized to spleen reveals IL-4 expression in hippocampus and cortex, abolished in IL-4 KO (n = 4 per group). Scale bars = 1 µm (A–G); 50 nm (J and K). Plots (I, N, and P) depict mean ± SEM. Statistics: Bayesian analysis, accuracy, * >80%, ** >90%, *** >95% (see Data S1). Source data are available for this figure: SourceData F4.

Fig 3: NCALD is enriched postnatally and in presynaptic terminals. (A) Western blot analysis of NCALD levels in brain lysates derived from the embryonic stage 16 (E16), postnatal day 1 (P1) and postnatal day 14 (P14) WT mice. Dots plots quantification reveals a 10-fold increase in the NCALD level in P10–14 brains; **P < 0.01; N.S. = not significant. (B) Representative confocal images of a WT brain immunostained for the NCALD, showing high protein expression in the forebrain and the midbrain and its abundance in the hippocampal and parahippocampal regions (magnified area); scale bar 500 μm. (C) Representative confocal images of cultured WT hippocampal neurons stained with NCALD antibody (green) and co-stained with synaptic markers VGLUT1 and VGAT (red), showing NCALD enrichment in the presynaptic terminals; scale bar 10 μm. Dotted white lines indicate areas taken for line plot analysis, where fluorescent signal for each channel is plotted relative to the distance. Asterisks in line plots represent the colocalization of NCALD with either VGLUT or VGAT. Uncropped Western blots are included in Supplementary Data Sheet 8.

Fig 4: Synapse number is reduced by Dnm1Ftfl/Ftfl, and accounts for reduced mPSC frequency. A, Images of biocytin-filled neurons (magenta) immunostained for vGlut1 (cyan), and vGAT (yellow). The top row shows representative excitatory neurons, and the bottom row shows representative inhibitory neurons. The left column shows representative WT neurons, and the right column shows representative Fitful neurons. Scale bars: 20 μm in length. B1–B4, Model effects tested using GEEs. C, Scatterplot showing excitatory synapse count (x-axis) and inhibitory synapse count (y-axis) onto excitatory neurons. Each dot represents an individual WT (black, N = 2, n = 11) or Fitful (chartreuse, N = 2, n = 15) neuron. Marginal boxplots define the median, IQR, and range along their respective axes. D, Plots showing synapse density (synapses per unit area) for the E-E (top) and I-E (bottom) motifs. WT swarm and box plots are in black and white, while Fitful swarm and box plots are colored for their respective motifs. Boxplots define the median, IQR, and range. E, Scatterplot showing excitatory synapse count (x-axis) and inhibitory synapse count (y-axis) onto inhibitory neurons. Each dot represents an individual WT (black, N = 2, n = 12) or Fitful (purple, N = 2, n = 13) neuron. Marginal boxplots define the median, IQR, and range along their respective axes. F, Plots showing synapse density (synapses per unit area) for the E-E (top) and I-E (bottom) motifs. WT swarm and box plots are in black and white, while Fitful swarm and box plots are colored for their respective motifs. Boxplots define the median, IQR, and range. G, Barplots showing the fraction of synapses located on the soma. Bars end at the mean, and error bars show the 95% confidence interval derived from bootstrap resampling. Dots represent individual neurons. H, Plots showing mPSC frequency normalized to synapse number for WT (white) and Fitful (colored) neurons; *pairwise comparison p < 0.05. For complete pairwise comparisons data, see Extended Data Figure 2-1.

Fig 5: AAV9-CamKIIa has little or no colocalization with VGluT2 and VGluT1 staining. (A) Top row: the infected cells with the CamKIIa promoter (AAV9) express mScarlet (RFP) and its overlap with VGluT2 (cyan), and second row: thresholded views of the top row and the heat map of RFP and VGluT2. (B) Top right grey box: fluorescent correlation plots of RFP and VGluT2 show very little co-expression. (C) First row: original images showing mScarlet (RFP), VGluT1 (green) and their overlap, and bottom row: the threshold views of the images and and the heat map for co-localization. (D) Bottom right grey box: the correlation for various levels of threshold appear dark. (N = 3 animals, n = 3–4 sections/animal). Scale bar 20 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\upmu$$\end{document}µm.